Search

Our previous study identified 8 risk and 9 protective plasma miRNAs associated with progression to end-stage kidney disease (ESKD) in diabetes. This study aimed to elucidate preanalytical factors that influence the quantification of circulating miRNAs. Using the EdgeSeq platform, which quantifies 2,002 miRNAs in plasma, including ESKD-associated miRNAs, we compared miRNA profiles in whole plasma versus miRNA profiles in RNA extracted from the same plasma specimens. Less than half of the miRNAs were detected in standard RNA extraction from plasma. Detection of individual and concentrations of miRNAs were much lower when RNA extracted from plasma was quantified by RNA sequencing (RNA-Seq) or quantitative reverse transcription PCR (qRT-PCR) platforms compared with EdgeSeq. Plasma profiles of miRNAs determined by the EdgeSeq platform had excellent reproducibility in assessment and had no variation with age, sex, hemoglobin A1c, BMI, and cryostorage time. The risk ESKD-associated miRNAs were detected and measured accurately only in whole plasma and using the EdgeSeq platform. Protective ESKD-associated miRNAs were detected by all platforms except qRT-PCR; however, correlations among concentrations obtained with different platforms were weak or nonexistent. In conclusion, preanalytical factors have a profound effect on detection and quantification of circulating miRNAs in ESKD in diabetes. Quantification of miRNAs in whole plasma and using the EdgeSeq platform may be the preferable method to study profiles of circulating cell-free miRNAs associated with ESKD and possibly other diseases.

Chronic kidney disease is a debilitating condition associated with significant morbidity and mortality. In recent years, the kidney effects of incretin-based therapies, particularly glucagon-like peptide-1 receptor agonists (GLP-1RAs), have garnered substantial interest in the management of type 2 diabetes and obesity. This review delves into the intricate interactions between the kidney, GLP-1RAs, and glucagon, shedding light on their mechanisms of action and potential kidney benefits. Both GLP-1 and glucagon, known for their opposing roles in regulating glucose homeostasis, improve systemic risk factors affecting the kidney, including adiposity, inflammation, oxidative stress, and endothelial function. Additionally, these hormones and their pharmaceutical mimetics may have a direct impact on the kidney. Clinical studies have provided evidence that incretins, including those incorporating glucagon receptor agonism, are likely to exhibit improved kidney outcomes. Although further research is necessary, receptor polypharmacology holds promise for preserving kidney function through eliciting vasodilatory effects, influencing volume and electrolyte handling, and improving systemic risk factors.

Current classification of chronic kidney disease (CKD) into stages using indirect systemic measures (estimated glomerular filtration rate (eGFR) and albuminuria) is agnostic to the heterogeneity of underlying molecular processes in the kidney thereby limiting precision medicine approaches. To generate a novel CKD categorization that directly reflects within kidney disease drivers we analyzed publicly available transcriptomic data from kidney biopsy tissue. A Self-Organizing Maps unsupervised artificial neural network machine-learning algorithm was used to stratify a total of 369 patients with CKD and 46 living kidney donors as healthy controls. Unbiased stratification of the discovery cohort resulted in identification of four novel molecular categories of disease termed CKD-Blue, CKD-Gold, CKD-Olive, CKD-Plum that were replicated in independent CKD and diabetic kidney disease datasets and can be further tested on any external data at kidneyclass.org. Each molecular category spanned across CKD stages and histopathological diagnoses and represented transcriptional activation of distinct biological pathways. Disease progression rates were highly significantly different between the molecular categories. CKD-Gold displayed rapid progression, with significant eGFR-adjusted Cox regression hazard ratio of 5.6 [1.01-31.3] for kidney failure and hazard ratio of 4.7 [1.3-16.5] for composite of kidney failure or a 40% or more eGFR decline. Urine proteomics revealed distinct patterns between the molecular categories, and a 25-protein signature was identified to distinguish CKD-Gold from other molecular categories. Thus, patient stratification based on kidney tissue omics offers a gateway to non-invasive biomarker-driven categorization and the potential for future clinical implementation, as a key step towards precision medicine in CKD., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objective: Tirzepatide reduces HbA1c and body weight, and creatinine-based estimated glomerular filtration rate (eGFR) decline. Unlike creatine-derived eGFR (eGFR-creatinine), cystatin C-derived eGFR (eGFR-cystatin C) is unaffected by muscle mass changes. We assessed effects of tirzepatide on eGFR-creatinine and eGFR-cystatin C., Research Design and Methods: Our primary outcome was eGFR change from baseline at 52 weeks with pooled tirzepatide (5, 10, and 15 mg) and titrated insulin glargine in adults with type 2 diabetes and high cardiovascular risk (SURPASS-4)., Results: Least squares mean (SE) eGFR-creatinine (mL/min/1.73 m2) changes from baseline with tirzepatide and insulin glargine were -2.5 (0.38) and -3.9 (0.38) (between-group difference, 1.4 [95% CI 0.3-2.4]) and -3.5 (0.37) and -5.3 (0.37) (between-group difference, 1.8 [95% CI 0.8-2.8]) for eGFR-cystatin C. Baseline, 1-year, and 1-year change from baseline values significantly correlated between eGFR-cystatin C and eGFR-creatinine. Measures of eGFR changes did not correlate with body weight changes., Conclusions: Tirzepatide slows the eGFR decline rate, supporting a kidney-protective effect., (© 2023 by the American Diabetes Association.)

Objective: The glucagon-like peptide-1 receptor agonist dulaglutide reduced MACE in the Researching Cardiovascular Events with a Weekly Incretin in Diabetes (REWIND) trial. This article expores the relationship of selected biomarkers to both dulaglutide and major adverse cardiovascular events (MACE)., Research Design and Methods: In this post hoc analysis, stored fasting baseline and 2-year plasma samples from 824 REWIND participants with MACE during follow-up and 845 matched non-MACE participants were analyzed for 2-year changes in 19 protein biomarkers. Two-year changes in 135 metabolites were also analyzed in 600 participants with MACE during follow-up and in 601 matched non-MACE participants. Linear and logistic regression models were used to identify proteins that were associated with both dulaglutide treatment and MACE. Similar models were used to identify metabolites that were associated with both dulaglutide treatment and MACE., Results: Compared with placebo, dulaglutide was associated with a greater reduction or lesser 2-year rise from baseline in N-terminal prohormone of brain natriuretic peptide (NT-proBNP), growth differentiation factor 15 (GDF-15), high-sensitivity C-reactive protein, and a greater 2-year rise in C-peptide. Compared with placebo, dulaglutide was also associated with a greater fall from baseline in 2-hydroxybutyric acid and a greater rise in threonine (P < 0.001). Increases from baseline in two of the proteins (but neither metabolite) were associated with MACE, including NT-proBNP (OR 1.267; 95% CI 1.119, 1.435; P < 0.001) and GDF-15 (OR 1.937; 95% CI 1.424, 2.634; P < 0.001)., Conclusions: Dulaglutide was associated with a reduced 2-year rise from baseline of NT-proBNP and GDF-15. Higher rises of these biomarkers were also associated with MACE., (© 2023 by the American Diabetes Association.)

Background: Chronic kidney disease (CKD) is a common comorbidity in people with diabetes mellitus, and a key risk factor for further life-threatening conditions such as cardiovascular disease. The early prediction of progression of CKD therefore is an important clinical goal, but remains difficult due to the multifaceted nature of the condition. We validated a set of established protein biomarkers for the prediction of trajectories of estimated glomerular filtration rate (eGFR) in people with moderately advanced chronic kidney disease and diabetes mellitus. Our aim was to discern which biomarkers associate with baseline eGFR or are important for the prediction of the future eGFR trajectory., Methods: We used Bayesian linear mixed models with weakly informative and shrinkage priors for clinical predictors (n = 12) and protein biomarkers (n = 19) to model eGFR trajectories in a retrospective cohort study of people with diabetes mellitus (n = 838) from the nationwide German Chronic Kidney Disease study. We used baseline eGFR to update the models' predictions, thereby assessing the importance of the predictors and improving predictive accuracy computed using repeated cross-validation., Results: The model combining clinical and protein predictors had higher predictive performance than a clinical only model, with an [Formula: see text] of 0.44 (95% credible interval 0.37-0.50) before, and 0.59 (95% credible interval 0.51-0.65) after updating by baseline eGFR, respectively. Only few predictors were sufficient to obtain comparable performance to the main model, with markers such as Tumor Necrosis Factor Receptor 1 and Receptor for Advanced Glycation Endproducts being associated with baseline eGFR, while Kidney Injury Molecule 1 and urine albumin-creatinine-ratio were predictive for future eGFR decline., Conclusions: Protein biomarkers only modestly improve predictive accuracy compared to clinical predictors alone. The different protein markers serve different roles for the prediction of longitudinal eGFR trajectories potentially reflecting their role in the disease pathway., (© 2023. The Author(s).)

Introduction: In the AWARD-7 study in patients with type 2 diabetes and moderate-to-severe chronic kidney disease, once-weekly dulaglutide slowed the decline in estimated glomerular filtration rate (eGFR) and decreased the urine albumin/creatinine ratio compared to insulin glargine at the end of 52 weeks of treatment. In this exploratory post hoc analysis, changes in two fibrosis biomarkers, serum PRO-C6 (type VI collagen formation) and urine C3M (type III collagen degradation), were evaluated., Methods: In the groups treated with dulaglutide 1.5 mg or insulin glargine (N = 330), serum PRO-C6 and urine C3M were measured using competitive enzyme-linked immunosorbent assays. Biomarker changes were assessed by a mixed-effects model for repeated measures. Pearson correlation analyses were conducted to determine associations between changes in kidney fibrosis biomarkers and eGFR measures at 52 weeks., Results: At weeks 26 and 52 of treatment in the overall population, serum PRO-C6 levels were significantly lower in the dulaglutide group versus insulin glargine group with percent change from baseline of (least squares mean ± standard error) -4.6% ± 1.9 and -0.2% ± 2.2 versus 5.7% ± 2.0 and 8.0% ± 2.3 (p < 0.01), respectively, and urine C3M levels were significantly higher in the dulaglutide group versus insulin glargine group with percent change from baseline of 10.9% ± 8.2 and 20.7% ± 8.8 versus -10.0% ± 6.5 and -16.9% ± 6.4 (p < 0.05), respectively. These findings appeared greater in the subgroup with macroalbuminuria. Serum PRO-C6 negatively correlated with eGFR, while urine C3M positively correlated with eGFR., Conclusion: Dulaglutide treatment was associated with biomarker changes that indicated lower type VI collagen formation and higher type III collagen degradation compared to treatment with insulin glargine, suggesting a potential drug effect to reduce kidney fibrosis., (© 2023 S. Karger AG, Basel.)

Background: In the SURPASS-4 trial, the dual GIP and GLP-1 receptor agonist tirzepatide reduced HbA 1c concentrations, bodyweight, and blood pressure more than titrated daily insulin glargine in people with type 2 diabetes inadequately controlled on oral diabetes treatments and with high cardiovascular risk. We aimed to compare the effects of tirzepatide and insulin glargine on kidney parameters and outcomes in people with type 2 diabetes., Methods: We did a post-hoc analysis of data from SURPASS-4, a randomised, open-label, parallel-group, phase 3 study at 187 sites (including private practice, research institutes, and hospitals) in 14 countries. Eligible participants were adults (age ≥18 years), with type 2 diabetes treated with any combination of metformin, sulfonylurea, or SGLT2 inhibitor, and with baseline HbA 1c of 7·5-10·5% (58-91 mmol/mol), BMI of 25 kg/m 2 or greater, and established cardiovascular disease or a high risk of cardiovascular events. Randomisation via an interactive web-response system was 1:1:1:3 to a once-weekly subcutaneous injection of tirzepatide (5 mg, 10 mg, or 15 mg) or a once-daily subcutaneous injection of titrated insulin glargine (100 U/mL). The study included up to 104 weeks of treatment, with a median treatment duration of 85 weeks. We compared the rates of estimated glomerular filtration rate (eGFR) decline and the urine albumin-creatinine ratio (UACR) between the combined tirzepatide groups and the insulin glargine group in the modified intention-to-treat population. The kidney composite outcome was time to first occurrence of eGFR decline of at least 40% from baseline, end-stage kidney disease, death owing to kidney failure, or new-onset macroalbuminuria. This study is registered with ClinicalTrials.gov, NCT03730662., Findings: Between Nov 20, 2018, and Dec 30, 2019, we screened 3045 people, of whom 1043 (34%) were ineligible, and 2002 (66%) were randomly assigned to a study drug (997 to tirzepatide and 1005 to insulin glargine). 1995 (>99%) of 2002 received at least one dose of tirzepatide (n=995) or insulin glargine (n=1000). At baseline, participants had a mean eGFR of 81·3 (SD 21·11) mL/min per 1·73 m 2 and a median UACR of 15·0 mg/g (IQR 5·0-55·8). The mean rate of eGFR decline was -1·4 (SE 0·2) mL/min per 1·73 m 2 per year in the combined tirzepatide groups and -3·6 (0·2) mL/min per 1·73 m 2 per year in the insulin group (between-group difference 2·2 [95% CI 1·6 to 2·8]). Compared with insulin glargine, the reduction in the annual rate of eGFR decline induced by tirzepatide was more pronounced in participants with eGFR less than 60 mL/min per 1·73 m 2 than in those with eGFR 60 mL/min per 1·73 m 2 or higher (between-group difference 3·7 [95% CI 2·4 to 5·1]). UACR increased from baseline to follow-up with insulin glargine (36·9% [95% CI 26·0 to 48·7]) but not with tirzepatide (-6·8% [-14·1 to 1·1]; between-group difference -31·9% [-37·7 to -25·7]). Participants who received tirzepatide showed a significantly lower occurrence of the composite kidney endpoint compared with those who received insulin glargine (hazard ratio 0·58 [95% CI 0·43 to 0·80])., Interpretation: Our analysis suggests that in people with type 2 diabetes and high cardiovascular risk, tirzepatide slowed the rate of eGFR decline and reduced UACR in clinically meaningful ways compared with insulin glargine., Funding: Eli Lilly and Company., Competing Interests: Declaration of interests HJLH has received grants or contracts from AstraZeneca, Boehringer Ingelheim, Janssen, and NovoNordisk; consulting fees from AstraZeneca, Bayer, Boehringer Ingelheim, CSL Behring, Chinook, Dimerix, Eli Lilly, Gilead, Goldfinch Bio, Janssen, Mitsubishi Tanabe Pharma, NovoNordisk, MSD, and Travere Therapeutics; payment or honoraria for speaking from AstraZeneca; and support for attending meetings from Eli Lilly. NS has received grants from AstraZeneca, Boehringer Ingelheim, Novartis, and Roche Diagnostics; and consulting fees from Afimmune, Amgen, AstraZeneca, Boehringer Ingelheim, Eli Lilly, Hanmi Pharmaceutical, MSD, Novartis, Novo Nordisk, Pfizer, and Sanofi. IP, AH, KLD, ZY, and RJW are employees and shareholders of Eli Lilly and Company. AH is a data monitoring committee board member for tirzepatide (Eli Lilly and Company). KRT has received grants or contracts from Bayer, Goldfinch Bio, and Travere; consulting fees from Eli Lilly, Boehringer Ingelheim, AstraZeneca, Goldfinch Bio, Travere Pharmaceuticals, Bayer, and Novo Nordisk; payment or honoraria for speaking from Eli Lilly, AstraZeneca, Bayer, and Novo Nordisk; has participated on data safety monitoring or advisory boards for the US National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health and the George Clinical Institute for Global Health; and reports a leadership or fiduciary role as Chair of the Diabetic Kidney Disease Collaborative Task Force, American Society of Nephrology. DZIC has received grants or contracts from Boehringer Ingelheim–Lilly, Merck, Janssen, Sanofi, AstraZeneca, CSL Behring, and Novo Nordisk; and consulting fees and speaking honoraria from Boehringer Ingelheim-Lilly, Merck, AstraZeneca, Sanofi, Mitsubishi-Tanabe, Abbvie, Janssen, Bayer, Prometic, BMS, Maze, Gilead, CSL Behring, Otsuka, Novartis, Yeungene, and NovoNordisk., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Circulating proteins associated with transforming growth factor-β (TGF-β) signaling are implicated in the development of diabetic kidney disease (DKD). It remains to be comprehensively examined which of these proteins are involved in the pathogenesis of DKD and its progression to end-stage kidney disease (ESKD) in humans. Using the SOMAscan proteomic platform, we measured concentrations of 25 TGF-β signaling family proteins in four different cohorts composed in total of 754 Caucasian or Pima Indian individuals with type 1 or type 2 diabetes. Of these 25 circulating proteins, we identified neuroblastoma suppressor of tumorigenicity 1 (NBL1, aliases DAN and DAND1), a small secreted protein known to inhibit members of the bone morphogenic protein family, to be most strongly and independently associated with progression to ESKD during 10-year follow-up in all cohorts. The extent of damage to podocytes and other glomerular structures measured morphometrically in 105 research kidney biopsies correlated strongly with circulating NBL1 concentrations. Also, in vitro exposure to NBL1 induced apoptosis in podocytes. In conclusion, circulating NBL1 may be involved in the disease process underlying progression to ESKD, and its concentration in circulation may identify subjects with diabetes at increased risk of progression to ESKD.

This study applies a large proteomics panel to search for new circulating biomarkers associated with progression to kidney failure in individuals with diabetic kidney disease. Four independent cohorts encompassing 754 individuals with type 1 and type 2 diabetes and early and late diabetic kidney disease were followed to ascertain progression to kidney failure. During ten years of follow-up, 227 of 754 individuals progressed to kidney failure. Using the SOMAscan proteomics platform, we measured baseline concentration of 1129 circulating proteins. In our previous publications, we analyzed 334 of these proteins that were members of specific candidate pathways involved in diabetic kidney disease and found 35 proteins strongly associated with risk of progression to kidney failure. Here, we examined the remaining 795 proteins using an untargeted approach. Of these remaining proteins, 11 were significantly associated with progression to kidney failure. Biological processes previously reported for these proteins were related to neuron development (DLL1, MATN2, NRX1B, KLK8, RTN4R and ROR1) and were implicated in the development of kidney fibrosis (LAYN, DLL1, MAPK11, MATN2, endostatin, and ROR1) in cellular and animal studies. Specific mechanisms that underlie involvement of these proteins in progression of diabetic kidney disease must be further investigated to assess their value as targets for kidney-protective therapies. Using multivariable LASSO regression analysis, five proteins (LAYN, ESAM, DLL1, MAPK11 and endostatin) were found independently associated with risk of progression to kidney failure. Thus, our study identified proteins that may be considered as new candidate prognostic biomarkers to predict risk of progression to kidney failure in diabetic kidney disease. Furthermore, three of these proteins (DLL1, ESAM, and MAPK11) were selected as candidate biomarkers when all SOMAscan results were evaluated., (Copyright © 2022 International Society of Nephrology. All rights reserved.)

Context: Tirzepatide substantially reduced hemoglobin A1c (HbA1c) and body weight in subjects with type 2 diabetes (T2D) compared with the glucagon-like peptide 1 receptor agonist dulaglutide. Improved glycemic control was associated with lower circulating triglycerides and lipoprotein markers and improved markers of beta-cell function and insulin resistance (IR), effects only partially attributable to weight loss., Objective: Assess plasma metabolome changes mediated by tirzepatide., Design: Phase 2b trial participants were randomly assigned to receive weekly subcutaneous tirzepatide, dulaglutide, or placebo for 26 weeks. Post hoc exploratory metabolomics and lipidomics analyses were performed., Setting: Post hoc analysis., Participants: 259 subjects with T2D., Intervention(s): Tirzepatide (1, 5, 10, 15 mg), dulaglutide (1.5 mg), or placebo., Main Outcome Measure(s): Changes in metabolite levels in response to tirzepatide were assessed against baseline levels, dulaglutide, and placebo using multiplicity correction., Results: At 26 weeks, a higher dose tirzepatide modulated a cluster of metabolites and lipids associated with IR, obesity, and future T2D risk. Branched-chain amino acids, direct catabolic products glutamate, 3-hydroxyisobutyrate, branched-chain ketoacids, and indirect byproducts such as 2-hydroxybutyrate decreased compared to baseline and placebo. Changes were significantly larger with tirzepatide compared with dulaglutide and directly proportional to reductions of HbA1c, homeostatic model assessment 2-IR indices, and proinsulin levels. Proportional to metabolite changes, triglycerides and diglycerides were lowered significantly compared to baseline, dulaglutide, and placebo, with a bias toward shorter and highly saturated species., Conclusions: Tirzepatide reduces body weight and improves glycemic control and uniquely modulates metabolites associated with T2D risk and metabolic dysregulation in a direction consistent with improved metabolic health., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In a phase 2 trial of once-weekly tirzepatide (1, 5, 10, or 15 mg), dulaglutide (1.5 mg), or placebo, the dual glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist tirzepatide dose-dependently reduced HbA1c and body weight in patients with type 2 diabetes. In this post hoc analysis, inflammation, endothelial dysfunction, and cellular stress biomarkers were measured at baseline, 4, 12, and 26 weeks to evaluate the additional effects of tirzepatide on cardiovascular risk factors. At 26 weeks, tirzepatide 10 and 15 mg decreased YKL-40 (also known as chitinase-3 like-protein-1), intercellular adhesion molecule 1 (ICAM-1), leptin, and growth differentiation factor 15 levels versus baseline, and YKL-40 and leptin levels versus placebo and dulaglutide. Tirzepatide 15 mg also decreased ICAM-1 levels versus placebo and dulaglutide, and high-sensitivity C-reactive protein (hsCRP) levels versus baseline and placebo, but not dulaglutide. GlycA, interleukin 6, vascular cell adhesion molecule 1, and N-terminal-pro hormone B-type natriuretic peptide levels were not significantly changed in any group. YKL-40, hsCRP, and ICAM-1 levels rapidly decreased within 4 weeks of treatment with tirzepatide 10 and 15 mg, whereas the decrease in leptin levels was more gradual and did not plateau by 26 weeks. In this hypothesis-generating exploratory analysis, tirzepatide decreased several biomarkers that have been associated with cardiovascular risk., (© 2021 Eli Lilly and Company. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Purpose: Currently, calculations of proton range in proton therapy patients are based on a conversion of CT Hounsfield units of patient tissues into proton relative stopping power. Uncertainties in this conversion necessitate larger proximal and distal planned target volume margins. Proton CT can potentially reduce these uncertainties by directly measuring proton stopping power. We aim to demonstrate proton CT imaging with complex porcine samples, to analyze in detail three-dimensional regions of interest, and to compare proton stopping powers directly measured by proton CT to those determined from x-ray CT scans., Methods: We have used a prototype proton imaging system with single proton tracking to acquire proton radiography and proton CT images of a sample of porcine pectoral girdle and ribs, and a pig's head. We also acquired close in time x-ray CT scans of the same samples and compared proton stopping power measurements from the two modalities. In the case of the pig's head, we obtained x-ray CT scans from two different scanners and compared results from high-dose and low-dose settings., Results: Comparing our reconstructed proton CT images with images derived from x-ray CT scans, we find agreement within 1% to 2% for soft tissues and discrepancies of up to 6% for compact bone. We also observed large discrepancies, up to 40%, for cavitated regions with mixed content of air, soft tissue, and bone, such as sinus cavities or tympanic bullae., Conclusions: Our images and findings from a clinically realistic proton CT scanner demonstrate the potential for proton CT to be used for low-dose treatment planning with reduced margins., (© 2021 American Association of Physicists in Medicine.)

The hepatokine follistatin is elevated in patients with type 2 diabetes (T2D) and promotes hyperglycemia in mice. Here we explore the relationship of plasma follistatin levels with incident T2D and mechanisms involved. Adjusted hazard ratio (HR) per standard deviation (SD) increase in follistatin levels for T2D is 1.24 (CI: 1.04-1.47, p < 0.05) during 19-year follow-up (n = 4060, Sweden); and 1.31 (CI: 1.09-1.58, p < 0.01) during 4-year follow-up (n = 883, Finland). High circulating follistatin associates with adipose tissue insulin resistance and non-alcoholic fatty liver disease (n = 210, Germany). In human adipocytes, follistatin dose-dependently increases free fatty acid release. In genome-wide association study (GWAS), variation in the glucokinase regulatory protein gene (GCKR) associates with plasma follistatin levels (n = 4239, Sweden; n = 885, UK, Italy and Sweden) and GCKR regulates follistatin secretion in hepatocytes in vitro. Our findings suggest that GCKR regulates follistatin secretion and that elevated circulating follistatin associates with an increased risk of T2D by inducing adipose tissue insulin resistance., (© 2021. The Author(s).)

Background: Mechanisms underlying the pro gression of diabetic kidney disease to ESKD are not fully understood., Methods: We performed global microRNA (miRNA) analysis on plasma from two cohorts consisting of 375 individuals with type 1 and type 2 diabetes with late diabetic kidney disease, and targeted proteomics analysis on plasma from four cohorts consisting of 746 individuals with late and early diabetic kidney disease. We examined structural lesions in kidney biopsy specimens from the 105 individuals with early diabetic kidney disease. Human umbilical vein endothelial cells were used to assess the effects of miRNA mimics or inhibitors on regulation of candidate proteins., Results: In the late diabetic kidney disease cohorts, we identified 17 circulating miRNAs, represented by four exemplars (miR-1287-5p, miR-197-5p, miR-339-5p, and miR-328-3p), that were strongly associated with 10-year risk of ESKD. These miRNAs targeted proteins in the axon guidance pathway. Circulating levels of six of these proteins-most notably, EFNA4 and EPHA2-were strongly associated with 10-year risk of ESKD in all cohorts. Furthermore, circulating levels of these proteins correlated with severity of structural lesions in kidney biopsy specimens. In contrast, expression levels of genes encoding these proteins had no apparent effects on the lesions. In in vitro experiments, mimics of miR-1287-5p and miR-197-5p and inhibitors of miR-339-5p and miR-328-3p upregulated concentrations of EPHA2 in either cell lysate, supernatant, or both., Conclusions: This study reveals novel mechanisms involved in progression to ESKD and points to the importance of systemic factors in the development of diabetic kidney disease. Some circulating miRNAs and axon guidance pathway proteins represent potential targets for new therapies to prevent and treat this condition., (Copyright © 2021 by the American Society of Nephrology.)

The functional interpretation of genome-wide association studies (GWAS) is challenging due to the cell-type-dependent influences of genetic variants. Here, we generated comprehensive maps of expression quantitative trait loci (eQTLs) for 659 microdissected human kidney samples and identified cell-type-eQTLs by mapping interactions between cell type abundances and genotypes. By partitioning heritability using stratified linkage disequilibrium score regression to integrate GWAS with single-cell RNA sequencing and single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing data, we prioritized proximal tubules for kidney function and endothelial cells and distal tubule segments for blood pressure pathogenesis. Bayesian colocalization analysis nominated more than 200 genes for kidney function and hypertension. Our study clarifies the mechanism of commonly used antihypertensive and renal-protective drugs and identifies drug repurposing opportunities for kidney disease., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Diabetic kidney disease (DKD) and its major clinical manifestation, progressive renal decline that leads to end-stage renal disease (ESRD), are a major health burden for individuals with diabetes. The disease process that underlies progressive renal decline comprises factors that increase risk as well as factors that protect against this outcome. Using untargeted proteomic profiling of circulating proteins from individuals in two independent cohorts with type 1 and type 2 diabetes and varying stages of DKD followed for 7 to 15 years, we identified three elevated plasma proteins-fibroblast growth factor 20 (OR, 0.69; 95% CI, 0.54 to 0.88), angiopoietin-1 (OR, 0.72; 95% CI, 0.57 to 0.91), and tumor necrosis factor ligand superfamily member 12 (OR, 0.75; 95% CI, 0.59 to 0.95)-that were associated with protection against progressive renal decline and progression to ESRD. The combined effect of these three protective proteins was demonstrated by very low cumulative risk of ESRD in those who had baseline concentrations above median for all three proteins, whereas the cumulative risk of ESRD was high in those with concentrations below median for these proteins at the beginning of follow-up. This protective effect was shown to be independent from circulating inflammatory proteins and clinical covariates and was confirmed in a third cohort of diabetic individuals with normal renal function. These three protective proteins may serve as biomarkers to stratify diabetic individuals according to risk of progression to ESRD and might also be investigated as potential therapeutics to delay or prevent the onset of ESRD., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose: Verification of patient-specific proton stopping powers obtained in the patient's treatment position can be used to reduce the distal and proximal margins needed in particle beam planning. Proton radiography can be used as a pretreatment instrument to verify integrated stopping power consistency with the treatment planning CT. Although a proton radiograph is a pixel by pixel representation of integrated stopping powers, the image may also be of high enough quality and contrast to be used for patient alignment. This investigation quantifies the accuracy and image quality of a prototype proton radiography system on a clinical proton delivery system., Methods: We have developed a clinical prototype proton radiography system designed for integration into efficient clinical workflows. We tested the images obtained by this system for water-equivalent thickness (WET) accuracy, image noise, and spatial resolution. We evaluated the WET accuracy by comparing the average WET and rms error in several regions of interest (ROI) on a proton radiograph of a custom peg phantom. We measured the spatial resolution on a CATPHAN Line Pair phantom and a custom edge phantom by measuring the 10% value of the modulation transfer function (MTF). In addition, we tested the ability to detect proton range errors due to anatomical changes in a patient with a customized CIRS pediatric head phantom and inserts of varying WET placed in the posterior fossae of the brain. We took proton radiographs of the phantom with each insert in place and created difference maps between the resulting images. Integrated proton range was measured from an ROI in the difference maps., Results: We measured the WET accuracy of the proton radiographic images to be ±0.2 mm (0.33%) from known values. The spatial resolution of the images was 0.6 lp/mm on the line pair phantom and 1.13 lp/mm on the edge phantom. We were able to detect anatomical changes producing changes in WET as low as 0.6 mm., Conclusion: The proton radiography system produces images with image quality sufficient for pretreatment range consistency verification., (© 2021 American Association of Physicists in Medicine.)

Introduction: Metabolic-associated fatty liver disease is common, with fibrosis the major determinant of adverse outcomes. Population-based screening tools with high diagnostic accuracy for the staging of fibrosis are lacking., Methods: Three independent cohorts, 2 with both liver biopsy and liver stiffness measurements (LSMs, n = 254 and 65) and a population sample (n = 713), were studied. The performance of a recently developed noninvasive algorithm (ADAPT [age, diabetes, PRO-C3 and platelets panel]) as well as aspartate aminotransferase-to-platelet ratio index, fibrosis-4, nonalcoholic fatty liver disease fibrosis score, and LSM was used to stage patients for significant (≥F2) and advanced (≥F3) fibrosis., Results: In the hospital-based cohorts, the N-terminal propeptide of type 3 collagen (Pro-C3) increased with fibrosis stage (P < 0.0001) and independently associated with advanced fibrosis (odds ratio = 1.091, 95% confidence interval [CI]: 1.053-1.113, P = 0.0001). ADAPT showed areas under the receiver operating characteristics curve of 0.831 (95% CI: 0.779-0.875) in the derivation and 0.879 (95% CI: 0.774-0.946) in the validation cohort for advanced fibrosis. This was superior to the existing fibrosis scores, aspartate aminotransferase-to-platelet ratio index, fibrosis-4, BARD (BMI, aspartate aminotransferase to alanine aminotransferase ratio [AAR], diabetes), and nonalcoholic fatty liver disease fibrosis score in most comparisons and comparable with LSM. Serial use of ADAPT and LSM had diagnostic accuracy of 92.5%, with 98% and 100% negative predictive value in the derivation and validation cohorts, respectively. In the population cohort, PRO-C3 associated with advanced fibrosis (P = 0.04), while ADAPT had a negative predictive value of 98% for excluding advanced fibrosis., Discussion: PRO-C3 and ADAPT reliably exclude advanced fibrosis in low-risk populations. The serial combination of ADAPT with LSM has high diagnostic accuracy with a low requirement for liver biopsy. The proposed algorithm would help stratify those who need biopsies and narrow down those patients who would need to be referred to specialty clinics., (Copyright © 2021 by The American College of Gastroenterology.)

Purpose: To demonstrate a proton-imaging system based on well-established fast scintillator technology to achieve high performance with low cost and complexity, with the potential of a straightforward translation into clinical use., Methods: The system tracks individual protons through one (X, Y) scintillating fiber tracker plane upstream and downstream of the object and into a 13-cm -thick scintillating block residual energy detector. The fibers in the tracker planes are multiplexed into silicon photomultipliers (SiPMs) to reduce the number of electronics channels. The light signal from the residual energy detector is collected by 16 photomultiplier tubes (PMTs). Only four signals from the PMTs are output from each event, which allows for fast signal readout. A robust calibration method of the PMT signal to residual energy has been developed to obtain accurate proton images. The development of patient-specific scan patterns using multiple input energies allows for an image to be produced with minimal excess dose delivered to the patient., Results: The calibration of signals in the energy detector produces accurate residual range measurements limited by intrinsic range straggling. We measured the water-equivalent thickness (WET) of a block of solid water (physical thickness of 6.10 mm) with a proton radiograph. The mean WET from all pixels in the block was 6.13 cm (SD 0.02 cm). The use of patient-specific scan patterns using multiple input energies enables imaging with a compact range detector., Conclusions: We have developed a prototype clinical proton radiography system for pretreatment imaging in proton radiation therapy. We have optimized the system for use with pencil beam scanning systems and have achieved a reduction of size and complexity compared to previous designs., (© 2020 American Association of Physicists in Medicine.)

Proton CT (pCT) is a promising new imaging technique that can reconstruct relative stopping power (RSP) more accurately than x-ray CT in each cubic millimeter voxel of the patient. This, in turn, will result in better proton range accuracy and, therefore, smaller planned tumor volumes (PTV). The hardware description and some reconstructed images have previously been reported. In a series of two contributions, we focus on presenting the software algorithms that convert pCT detector data to the final reconstructed pCT images for application in proton treatment planning. There were several options on how to accomplish this, and we will describe our solutions at each stage of the data processing chain. In the first paper of this series, we present the data acquisition with the pCT tracking and energy-range detectors and how the data are preprocessed, including the conversion to the well-formatted track information from tracking data and water-equivalent path length from the data of a calibrated multi-stage energy-range detector. These preprocessed data are then used for the initial image formation with an FDK cone-beam CT algorithm. The output of data acquisition, preprocessing, and FDK reconstruction is presented along with illustrative imaging results for two phantoms, including a pediatric head phantom. The second paper in this series will demonstrate the use of iterative solvers in conjunction with the superiorization methodology to further improve the images resulting from the upfront FDK image reconstruction and the implementation of these algorithms on a hybrid CPU/GPU computer cluster.

Aim: To better understand the marked decrease in serum triglycerides observed with tirzepatide in patients with type 2 diabetes, additional lipoprotein-related biomarkers were measured post hoc in available samples from the same study., Materials and Methods: Patients were randomized to receive once-weekly subcutaneous tirzepatide (1, 5, 10 or 15 mg), dulaglutide (1.5 mg) or placebo. Serum lipoprotein profile, apolipoprotein (apo) A-I, B and C-III and preheparin lipoprotein lipase (LPL) were measured at baseline and at 4, 12 and 26 weeks. Lipoprotein particle profile by nuclear magnetic resonance was assessed at baseline and 26 weeks. The lipoprotein insulin resistance (LPIR) score was calculated., Results: At 26 weeks, tirzepatide dose-dependently decreased apoB and apoC-III levels, and increased serum preheparin LPL compared with placebo. Tirzepatide 10 and 15 mg decreased large triglyceride-rich lipoprotein particles (TRLP), small low-density lipoprotein particles (LDLP) and LPIR score compared with both placebo and dulaglutide. Treatment with dulaglutide also reduced apoB and apoC-III levels but had no effect on either serum LPL or large TRLP, small LDLP and LPIR score. The number of total LDLP was also decreased with tirzepatide 10 and 15 mg compared with placebo. A greater reduction in apoC-III with tirzepatide was observed in patients with high compared with normal baseline triglycerides. At 26 weeks, change in apoC-III, but not body weight, was the best predictor of changes in triglycerides with tirzepatide, explaining up to 22.9% of their variability., Conclusions: Tirzepatide treatment dose-dependently decreased levels of apoC-III and apoB and the number of large TRLP and small LDLP, suggesting a net improvement in atherogenic lipoprotein profile., (© 2020 Eli Lilly and Company. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Objective: The role of fibrosis in early progressive renal decline in type 2 diabetes is unknown. Circulating WFDC2 (WAP four-disulfide core domain protein 2) and matrix metalloproteinase 7 (MMP-7; Matrilysin) are postulated to be biomarkers of renal fibrosis. This study examined an association of circulating levels of these proteins with early progressive renal decline., Research Design and Methods: Individuals with type 2 diabetes enrolled in the Joslin Kidney Study with an estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m 2 were monitored for 6-12 years to ascertain fast early progressive renal decline, defined as eGFR loss ≥5 mL/min/1.73 m 2 /year., Results: A total of 1,181 individuals were studied: 681 without and 500 with albuminuria. Median eGFR and albumin-to-creatinine ratio (ACR) at baseline were 97 mL/min/1.73 m 2 and 24 mg/g, respectively. During follow-up, 152 individuals experienced fast early progressive renal decline: 6.9% in those with normoalbuminuria and 21% with albuminuria. In both subgroups, the risk of renal decline increased with increasing baseline levels of WFDC2 ( P < 0.0001) and MMP-7 ( P < 0.0001). After adjustment for relevant clinical characteristics and known biomarkers, an increase by one quartile in the fibrosis index (combination of levels of WFDC2 and MMP-7) was associated with higher risk of renal decline (odds ratio 1.63; 95% CI 1.30-2.04). The association was similar and statistically significant among patients with and without albuminuria., Conclusions: Elevation of circulating profibrotic proteins is associated with the development of early progressive renal decline in type 2 diabetes. This association is independent from albuminuria status and points to the importance of the fibrotic process in the development of early renal decline., (© 2020 by the American Diabetes Association.)

Objective: To determine the effect of tirzepatide, a dual agonist of glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 receptors, on biomarkers of nonalcoholic steatohepatitis (NASH) and fibrosis in patients with type 2 diabetes mellitus (T2DM)., Research Design and Methods: Patients with T2DM received either once weekly tirzepatide (1, 5, 10, or 15 mg), dulaglutide (1.5 mg), or placebo for 26 weeks. Changes from baseline in alanine aminotransferase (ALT), aspartate aminotransferase (AST), keratin-18 (K-18), procollagen III (Pro-C3), and adiponectin were analyzed in a modified intention-to-treat population., Results: Significant ( P < 0.05) reductions from baseline in ALT (all groups), AST (all groups except tirzepatide 10 mg), K-18 (tirzepatide 5, 10, 15 mg), and Pro-C3 (tirzepatide 15 mg) were observed at 26 weeks. Decreases with tirzepatide were significant compared with placebo for K-18 (10 mg) and Pro-C3 (15 mg) and with dulaglutide for ALT (10, 15 mg). Adiponectin significantly increased from baseline with tirzepatide compared with placebo (10, 15 mg)., Conclusions: In post hoc analyses, higher tirzepatide doses significantly decreased NASH-related biomarkers and increased adiponectin in patients with T2DM., Competing Interests: This article is featured in a podcast available at https://www.diabetesjournals.org/content/diabetes-core-update-podcasts., (© 2020 by the American Diabetes Association.)

Clinical risk factors explain only a fraction of the variability of estimated glomerular filtration rate (eGFR) decline in people with type 2 diabetes. Cross-omics technologies by virtue of a wide spectrum screening of plasma samples have the potential to identify biomarkers for the refinement of prognosis in addition to clinical variables. Here we utilized proteomics, metabolomics and lipidomics panel assay measurements in baseline plasma samples from the multinational PROVALID study (PROspective cohort study in patients with type 2 diabetes mellitus for VALIDation of biomarkers) of patients with incident or early chronic kidney disease (median follow-up 35 months, median baseline eGFR 84 mL/min/1.73 m 2 , urine albumin-to-creatinine ratio 8.1 mg/g). In an accelerated case-control study, 258 individuals with a stable eGFR course (median eGFR change 0.1 mL/min/year) were compared to 223 individuals with a rapid eGFR decline (median eGFR decline -6.75 mL/min/year) using Bayesian multivariable logistic regression models to assess the discrimination of eGFR trajectories. The analysis included 402 candidate predictors and showed two protein markers (KIM-1, NTproBNP) to be relevant predictors of the eGFR trajectory with baseline eGFR being an important clinical covariate. The inclusion of metabolomic and lipidomic platforms did not improve discrimination substantially. Predictions using all available variables were statistically indistinguishable from predictions using only KIM-1 and baseline eGFR (area under the receiver operating characteristic curve 0.63). Thus, the discrimination of eGFR trajectories in patients with incident or early diabetic kidney disease and maintained baseline eGFR was modest and the protein marker KIM-1 was the most important predictor., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The genomic regulatory networks underlying the pathogenesis of non-ST-segment elevation acute coronary syndrome (NSTE-ACS) are incompletely understood. As intermediate traits, protein biomarkers report on underlying disease severity and prognosis in NSTE-ACS. We hypothesized that integration of dense microRNA (miRNA) profiling with biomarker measurements would highlight potential regulatory pathways that underlie the relationships between prognostic biomarkers, miRNAs, and cardiovascular phenotypes. We performed miRNA sequencing using whole blood from 186 patients from the TRILOGY-ACS trial. Seven circulating prognostic biomarkers were measured: NH 2 -terminal pro-B-type natriuretic peptide (NT-proBNP), high-sensitivity C-reactive protein, osteopontin (OPN), myeloperoxidase, growth differentiation factor 15, monocyte chemoattractant protein, and neopterin. We tested miRNAs for association with each biomarker with generalized linear models and controlled the false discovery rate at 0.05. Ten miRNAs, including known cardiac-related miRNAs 25-3p and 423-3p, were associated with NT-proBNP levels (min. P = 7.5 × 10 -4 ) and 48 miRNAs, including cardiac-related miRNAs 378a-3p, 20b-5p and 320a, -b, and -d, were associated with OPN levels (min. P = 1.6 × 10 -6 ). NT-proBNP and OPN were also associated with time to cardiovascular death, myocardial infarction (MI), or stroke in the sample. By integrating large-scale miRNA profiling with circulating biomarkers as intermediate traits, we identified associations of known cardiac-related and novel miRNAs with two prognostic biomarkers and identified potential genomic networks regulating these biomarkers. These results, highlighting plausible biological pathways connecting miRNAs with biomarkers and outcomes, may inform future studies seeking to delineate genomic pathways underlying NSTE-ACS outcomes.

Objective: Proton beam therapy is an emerging modality for cancer treatment that, compared to X-ray radiation therapy, promises to provide better dose delivery to clinical targets with lower doses to normal tissues. Crucial to accurate treatment planning and dose delivery is knowledge of the water equivalent path length (WEPL) of each ray, or pencil beam, from the skin to every point in the target. For protons, this length is estimated from relative stopping power based on X-ray Hounsfield units. Unfortunately, such estimates lead to 3 to 4% uncertainties in the proton range prediction. Therefore, protons in the Bragg peak may overshoot (or undershoot) the desired stopping depth in the target causing tissue damage beyond the target volume. Recent studies indicate that tomographic imaging using protons has the potential to provide directly more accurate measurement of RSPs with significantly lower radiation dose than X-rays. We are currently working on a proton radiography system that promises to provide accurate two-dimensional (2D) images of WEPL values for protons that pass through the body. These will be suitable for positioning and range verification in daily treatments. In this study, we demonstrate that this system is capable of rapidly achieving such accurate images in clinically meaningful times., Methods: We have developed a software platform to characterize the potential performance of the prototype proton radiography system. We use Geant4 to simulate raw data detected by the device. An especially-written software - pRad - was written to process these data as they are received and uses iterative methods to generate radiographs. The software has been designed to generate a radiograph from a few million protons in under a minute after receiving the first proton from the device. We used a head phantom with known chemical compositions that could be modelled quite accurately in Geant4 simulations of proton radiographs. The radiographs are displayed as pixelated WEPL values displayed on a 2D gray scale image of WEPL values., Results: Rapid radiograph reconstruction of 3D phantoms using simulated proton pencil beams have been achieved with our software platform. On a modest desktop computer with a single central processing unit (CPU) and a single graphics processing unit (GPU), it takes about 11 seconds to reconstruct images using iterative linear algorithms to reconstruct a radiograph from 7.6 million protons. For the radiographic reconstructions of the head phantom described here, the mean WEPL errors, in the proton radiograph using a large majority of the pixels in the complete image were less than 1 mm when compared to images obtained without proton scattering and without detector resolution included., Conclusion: We have demonstrated, through computer simulations of proton irradiation of a pediatric head phantom using the newly built pRad detector and image reconstruction software, that high quality proton radiographs can be generated for patient alignment and verification of water equivalent thickness of the patient before each treatment., Competing Interests: Conflict of Interest Statement The authors have intellectual property rights to the innovations described in this paper. James S. Welsh has served as a medical advisor to ProTom International. Don F. Dejongh is a co-owner of ProtonVDA Inc.

Chronic inflammation is postulated to be involved in the development of end-stage renal disease in diabetes, but which specific circulating inflammatory proteins contribute to this risk remain unknown. To study this, we examined 194 circulating inflammatory proteins in subjects from three independent cohorts with type 1 and type 2 diabetes. In each cohort, we identified an extremely robust kidney risk inflammatory signature (KRIS), consisting of 17 proteins enriched in tumor necrosis factor-receptor superfamily members, that was associated with a 10-year risk of end-stage renal disease. All these proteins had a systemic, non-kidney source. Our prospective study findings provide strong evidence that KRIS proteins contribute to the inflammatory process underlying end-stage renal disease development in both types of diabetes. These proteins point to new therapeutic targets and new prognostic tests to identify subjects at risk of end-stage renal disease, as well as biomarkers to measure responses to treatment of diabetic kidney disease.

Growth/differentiation factor-15 (GDF-15) is a distant member of the transforming growth factor β (TGF-β) superfamily and is widely expressed in multiple mammalian tissues. Its expression is highly regulated and is often induced in response to conditions associated with cellular stress. GDF15 serum levels have a strong association with many diseases, including inflammation, cancer, cardiovascular diseases, and obesity, and potentially serve as reliable predictor of disease progression. A functional role for GDF15 has been suggested in cancer, cardiovascular disease, kidney disease and metabolic disease. However, the knowledge of its pathophysiological function at the molecular level is still limited and requires more investigation. Recent identification of the endogenous receptor for GDF15 may provide additional insight in to its' molecular mechanisms and relationship to disease states.

Background: Inflammation signaled by Janus kinases (JAKs) promotes progression of diabetic kidney disease (DKD). Baricitinib is an oral, reversible, selective inhibitor of JAK1 and JAK2. This study tested the efficacy of baricitinib versus placebo on albuminuria in adults with Type 2 diabetes at high risk for progressive DKD., Methods: In this Phase 2, double-blind, dose-ranging study, participants were randomized 1:1:1:1:1 to receive placebo or baricitinib (0.75 mg daily; 0.75 mg twice daily; 1.5 mg daily; or 4 mg daily), for 24 weeks followed by 4-8 weeks of washout., Results: Participants (N = 129) were 63±9.1 (mean±standard deviation) years of age, 27.1% (35/129) women and 11.6% (15/129) African-American race. Baseline hemoglobin A1c (HbA1c) was 7.3±1% and estimated glomerular filtration rate was 45.0±12.1 mL/min/1.73 m2 with first morning urine albumin-creatinine ratio (UACR) of 820 (407-1632) (median; interquartile range) mg/g. Baricitinib, 4 mg daily, decreased morning UACR by 41% at Week 24 compared with placebo (ratio to baseline 0.59, 95% confidence interval 0.38-0.93, P = 0.022). UACR was decreased at Weeks 12 and 24 and after 4-8 weeks of washout. Baricitinib 4 mg decreased inflammatory biomarkers over 24 weeks (urine C-X-C motif chemokine 10 and urine C-C motif ligand 2, plasma soluble tumor necrosis factor receptors 1 and 2, intercellular adhesion molecule 1 and serum amyloid A). The only adverse event rate that differed between groups was anemia at 32.0% (8/25) for baricitinib 4 mg daily versus 3.7% (1/27) for placebo., Conclusions: Baricitinib decreased albuminuria in participants with Type 2 diabetes and DKD. Further research is required to determine if baricitinib reduces DKD progression.

Objective: The decline of estimated glomerular filtration rate (eGFR) in patients with type 2 diabetes is variable, and early interventions would likely be cost-effective. We elucidated the contribution of 17 plasma biomarkers to the prediction of eGFR loss on top of clinical risk factors., Research Design and Methods: We studied participants in PROVALID (PROspective cohort study in patients with type 2 diabetes mellitus for VALIDation of biomarkers), a prospective multinational cohort study of patients with type 2 diabetes and a follow-up of more than 24 months ( n = 2,560; baseline median eGFR, 84 mL/min/1.73 m 2 ; urine albumin-to-creatinine ratio, 8.1 mg/g). The 17 biomarkers were measured at baseline in 481 samples using Luminex and ELISA. The prediction of eGFR decline was evaluated by linear mixed modeling., Results: In univariable analyses, 9 of the 17 markers showed significant differences in median concentration between stable and fast-progressing patients. A linear mixed model for eGFR obtained by variable selection exhibited an adjusted R 2 of 62%. A panel of 12 biomarkers was selected by the procedure and accounted for 34% of the total explained variability, of which 32% was due to 5 markers. The individual contribution of each biomarker to the prediction of eGFR decline on top of clinical predictors was generally low. When included into the model, baseline eGFR exhibited the largest explained variability of eGFR decline ( R 2 of 79%), and the contribution of each biomarker dropped below 1%., Conclusions: In this longitudinal study of patients with type 2 diabetes and maintained eGFR at baseline, 12 of the 17 candidate biomarkers were associated with eGFR decline, but their predictive power was low., (© 2018 by the American Diabetes Association.)

To identify determinants of early progressive renal decline in type 2 diabetes a range of markers was studied in 1032 patients enrolled into the 2nd Joslin Kidney Study. eGFR slopes estimated from serial measurements of serum creatinine during 5-12 years of follow-up were used to define early renal decline. At enrollment, all patients had normal eGFR, 58% had normoalbuminuria and 42% had albuminuria. Early renal decline developed in 6% and in 18% patients, respectively. As determinants, we examined baseline values of clinical characteristics, circulating markers: TNFR1, KIM-1, and FGF23, and urinary markers: albumin, KIM-1, NGAL, MCP-1, EGF (all normalized to urinary creatinine) and the ratio of EGF to MCP-1. In univariate analysis, all plasma and urinary markers were significantly associated with risk of early renal decline. When analyzed together, systolic blood pressure, TNFR1, KIM-1, the albumin to creatinine ratio, and the EGF/MCP-1 ratio remained significant with the latter having the strongest effect. Integration of these markers into a multi-marker prognostic test resulted in a significant improvement of discriminatory performance of risk prediction of early renal decline, compared with the albumin to creatinine ratio and systolic blood pressure alone. However, the positive predictive value was only 50% in albuminuric patients. Thus, markers in plasma and urine indicate that the early progressive renal decline in Type 2 diabetes has multiple determinants with strong evidence for involvement of tubular damage. However, new, more informative markers are needed to develop a better prognostic test for such decline that can be used in a clinical setting., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Design of Phase III trials for diabetic nephropathy currently requires patients at a high risk of progression defined as within three years of a hard end point (end-stage renal disease, 40% loss of estimated glomerular filtration rate, or death). To improve the design of these trials, we used natural history data from the Joslin Kidney Studies of chronic kidney disease in patients with diabetes to develop an improved criterion to identify such patients. This included a training cohort of 279 patients with type 1 diabetes and 134 end points within three years, and a validation cohort of 221 patients with type 2 diabetes and 88 end points. Previous trials selected patients using clinical criteria for baseline urinary albumin-to-creatinine ratio and estimated glomerular filtration rate. Application of these criteria to our cohort data yielded sensitivities (detection of patients at risk) of 70-80% and prognostic values of only 52-63%. We applied classification and regression trees analysis to select from among all clinical characteristics and markers the optimal prognostic criterion that divided patients with type 1 diabetes according to risk. The optimal criterion was a serum tumor necrosis factor receptor 1 level over 4.3 ng/ml alone or 2.9-4.3 ng/ml with an albumin-to-creatinine ratio over 1900 mg/g. Remarkably, this criterion produced similar results in both type 1 and type 2 diabetic patients. Overall, sensitivity and prognostic value were high (72% and 81%, respectively). Thus, application of this criterion to enrollment in future clinical trials could reduce the sample size required to achieve adequate statistical power for detection of treatment benefits., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Diabetic nephropathy imposes a substantial cardiovascular and renal burden contributing to both morbidity and excess mortality. Progression of chronic kidney disease (CKD) in diabetes mellitus is variable, and few biomarkers are available to predict progression accurately. Identification of novel predictive biomarkers may inform clinical care and assist in the design of clinical trials. We hypothesized that urinary and plasma protein biomarkers predict CKD progression independently of the known clinical markers such as albuminuria and estimated glomerular filtration rate (eGFR) in diabetic nephropathy., Methods: We studied 67 US veterans with CKD due to type 2 diabetes mellitus and 20 age-matched controls (no CKD, hypertension or cardiovascular disease). After clinical evaluation and the collection of blood and urine specimens for 24 biomarkers, we followed subjects prospectively for the next 2-6 years. CKD progression was defined in three ways: (i) clinically by examining eGFR versus time plots for each individual (slope progression), (ii) progression to end-stage renal disease (ESRD) and (iii) a composite outcome of ESRD or death., Results: Among 17 urinary and 7 plasma biomarkers evaluated, the relationship of the biomarkers with outcome was as follows: (i) for progression identified by eGFR plots, urinary C-terminal fibroblast growth factor (FGF)-23 emerged to have the strongest primary association (adjusted odds ratio [aOR] 2.08, P = 0.008); (ii) for ESRD, plasma vascular endothelial growth factor (VEGF) had an association (aOR: 1.44, P = 0.027) and (iii) for the composite outcome of death and ESRD, plasma C-terminal FGF-23 also had a robust direct association (aOR: 3.07, P = 0.008)., Conclusion: The relationship of biomarkers with future progression of CKD is complex and depends in part on how CKD progression is defined. Biomarkers in the FGF-23 and VEGF-A pathways predicted patient progression independently of albuminuria levels in this patient cohort. Additional studies in other cohorts will help further validate this pilot study., (Published by Oxford University Press on behalf of ERA-EDTA 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Objective: Alzheimer's disease (AD) is a devastating neurological disease characterized by pathological proteolytic cleavage of tau protein, which appears to initiate death of the neurons. The objective of this study was to investigate whether a proteolytic fragment of the tau protein could serve as blood-based biomarker of cognitive function in AD., Methods: We developed a highly sensitive ELISA assay specifically detecting an A Disintegrin and Metalloproteinase 10 (ADAM10)-generated fragment of tau (Tau-A). We characterized the assay in detail with to respect specificity and reactivity in healthy human serum. We used samples from the Tg4510 tau transgenic mice, which over-express the tau mutant P301L and exhibit a tauopathy with similarities to that observed in AD. We used serum samples from 21 well-characterized Alzheimer's patients, and we correlated the Tau-A levels to cognitive function., Results: The Tau-A ELISA specifically detected the cleavage sequence at the N-terminus of a fragment of tau generated by ADAM10 with no cross-reactivity to intact tau or brain extracts. In brain extracts from Tg4510 mice compared to wt controls we found 10-fold higher levels of Tau-A (p<0.001), which indicates a pathological relevance of this marker. In serum from healthy individuals we found robust and reproducible levels of Tau-A, indicating that the analyte is present in serum. In serum from AD patients an inverse correlation (R² = 0.46, p<0.001) between the cognitive assessment score (Mattis Dementia Rating Scale (MDRS)) and Tau-A levels was observed., Conclusion: Based on the hypothesis that tau is cleaved proteolytically and then released into the blood, we here provide evidence for the presence of an ADAM10-generated tau fragment (Tau-A) in serum. In addition, the levels of Tau-A showed an inverse correlation to cognitive function, which could indicate that this marker is a serum marker with pathological relevance for AD.

Background: Titin is a muscle-specific protein found in cardiac and skeletal muscles which is responsible for restoring passive tension. Levels and functioning of titin have been shown to be affected by cardiac damage. Due to the inherent difficulty of measuring titin levels in vivo in a clinical setting, we aimed to develop an assay that could reliably measure fragments of degraded titin in serum and potentially be used in the assessment of cardiac muscle damage., Methods: A competitive ELISA was developed to specifically measure levels of the titin sequence 12670' NVTVEARLIK 12679', derived by the degradation of titin by matrix metalloproteinase (MMP)-12. Serum samples from 90 individuals were divided into 3 equally sized groups. One group had been diagnosed with acute myocardial infarction (AMI) while the remaining two were asymptomatic individuals either with CT-scan signs of coronary calcium (CT-plusCa) or without coronary calcium (CT-noCa)., Results: Mean geometric levels of the titin fragment in the CT-noCa group were 506.5 ng/ml (± 43.88). The CT-plusCa group showed 50.6% higher levels of the marker [763 ng/ml (± 90.14)] (P < 0.05). AMI patients showed 56.3% higher levels [792 ng/ml (± 149)] (P < 0.05)., Conclusions: The titin-12670 fragment is present in both individuals with undiagnosed and diagnosed CVD. The statistically significant increase in level of the marker in the AMI group is indicative that this neoepitope biomarker may be a useful serological marker in AMI.

Aim: To investigate whether increased levels of vimentin citrullinated peptides identified by MS in articular cartilage can be measured in pathologies other than rheumatoid arthritis and be utilised for diagnostic purposes., Methods: A monoclonal antibody against the sequence RLRSSVPGV-citrulline (VICM) was developed and evaluated in a carbon tetrachloride (CCl(4)) (n=52 + 28 controls) rat model of liver fibrosis and two clinical cohorts of adult patients with hepatitis C (HCV) (n=92) and non-alcoholic fatty liver disease (NAFLD) (n=62), and compared to healthy controls., Results: In CCl(4)-treated rats, mean systemic VICM levels increased 31% at week 12 (176 ng/mL, P<0.001), 41.7% at weeks 16 (190 ng/mL, P<0.001), 49.2% at weeks 20 (200 ng/ml, P<0.001), compared to controls (134 ng/mL). VICM levels correlated with total hepatic collagen determined by Sirius red staining of rat livers (r=0.75, P<0.05). In the HCV cohort, when stratified according to the METAVIR F score, VICM levels were 63% higher in F0 (632 ng/mL ±75, p<0.05), 54% in F1 (597 ng/mL ±41.3, p<0.05) and 62% in F2 (628 ng/mL ±59, p<0.05) all compared to controls. In the NAFLD cohort, VICM levels were 20.6% higher in F0 (339 ±12 ng/mL, P<0.05), 23.8% in F1 (348 ±12 ng/mL, P<0.05) and 28.8% in F2 (362 ±25 P<0.05)., Conclusion: We demonstrated increased serological levels of citrullinated and MMP degraded vimentin in an animal model of liver fibrosis and in early fibrosis associated with HCV and NAFLD patients. These data suggest that citrullinated and MMP degraded proteins are also present in liver fibrosis.

Cuprizone intoxication is a commonly used model of demyelination that allows the temporal separation of demyelination and remyelination. The underlying biochemical alterations leading to demyelination, using this model, remain unclear and may be multifold. Analysis of proteomic changes within the brains of cuprizone-exposed animals may help elucidate key cellular processes. In the current study, we report the results of the liquid chromatography tandem mass spectrometry analysis of total protein from the brain hemispheres of control and toxin-exposed mice at 6 weeks of exposure and after 3 and 6 weeks of recovery to identify protein changes during the remyelination phase. We found that at 6 weeks of cuprizone exposure, myelin proteins were reduced compared to controls and increased throughout the course of recovery, as expected. In contrast, other protein groups, such as proteins related to mitochondrial function, were increased at 6 weeks of treatment compared to untreated controls and returned toward control levels following withdrawal of toxin. These results suggest that a global proteomic analysis of the brain tissue of cuprizone-treated mice can identify changes related to the demyelination/remyelination process.

Objective: To develop a short-term in vivo model in rats, with an enzyme-linked immunosorbent assay (ELISA) readout for specific aggrecanase-cleaved aggrecan fragments, to facilitate testing of aggrecanase inhibitors., Methods: Monosodium iodoacetate (MIA), a metabolic inhibitor, was injected into the right knee joint of male Lewis rats and the release of aggrecanase-cleaved fragments of aggrecan containing the NITEGE or ARGN neoepitope was measured in the synovial fluid at 7 days post MIA injection using novel ELISAs. The ELISAs utilize a commercial antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan, in combination with either an alpha-NITEGE antibody (NITEGE ELISA) or an alpha-ARGS/BC3 antibody (ARGS ELISA), to detect aggrecanase-cleavage of aggrecan within the interglobular domain (IGD). Aggrecan fragments present in in vitro digests, in cytokine-treated cartilage explant culture supernatants and in rat synovial fluid lavage samples were detected and quantified using the two ELISAs. Small molecule inhibitors of aggrecanase activity were dosed orally on days 3-7 to determine their ability to inhibit MIA-induced generation of the NITEGE and ARGN neoepitopes measured in the rat synovial fluid., Results: The NITEGE assay was shown to specifically detect the N-terminal fragment of aggrecan comprising the G1 domain and the NITEGE neoepitope sequence. This assay can readily measure aggrecanase-cleaved bovine, human and rat aggrecan without the need for deglycosylation. The ARGS assay specifically detects C-terminal fragments of aggrecan comprising the ARGS/ARGN neoepitope and the G2 domain. Keratan sulfate (KS) residues of aggrecan interfere with this ELISA, and hence this assay works well with native rat articular cartilage aggrecan (that lacks KS residues) and with deglycosylated bovine and human aggrecan. Injection of MIA into the rat knee joints resulted in a time-dependent increase in the release of aggrecanase-cleaved aggrecan fragments into the synovial fluid and treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of the generation of these neoepitopes., Conclusions: We have established a short-term in vivo model in rats that involves measurement of synovial fluid biomarkers that are dependent on aggrecanase activity in the joint. The short duration of the model combined with the mechanistic biomarker readout makes it very useful for the initial in vivo screening of aggrecanase inhibitors prior to testing them in time and resource-intensive disease models of osteoarthritis (OA)., (Copyright 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA., Methods: The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA., Results: The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity., Conclusions: We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA., (Copyright 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: To identify, characterize, and compare proteolysis peptide products generated by metalloprotease digests of human articular cartilage., Methods: Human articular cartilage was digested by the addition of exogenous metalloproteases, including matrix metalloproteinases 2, 3, 8, 9, 12, and 13 and aggrecanases ADAMTS-4 and ADAMTS-5. Proteolyzed peptide products were identified by proteomics methods using mass spectrometry., Results: Complete sequences of the peptides proteolyzed from human articular cartilage, including N- and C-termini and hydroxylated posttranslational modifications, were determined. A wide variety of peptides, originating from types I, II, and III collagen, biglycan, prolargin, fibromodulin, fibronectin, decorin, cartilage oligomeric matrix protein, cartilage intermediate-layer protein, megakaryocyte-stimulating factor, mimecan, aggrecan, and lumican, was analyzed following metalloprotease digestion. Release of peptides varied as a function of time, enzyme specificity, and abundance. Specific type II collagen peptide biomarkers, including those containing the three-quarter-length fragment cleavage site and those containing the domains for helical peptide of type II collagen and C-telopeptide of type II collagen, were observed after release by selected proteases., Conclusion: The use of intact cartilage instead of purified protein substrates in the assay allowed for the identification of novel potential substrates and cleavage sites for individual enzymes under more physiologically relevant conditions. Characterization of these cartilage matrix peptides may help in the development of pharmacodynamic biomarkers of cartilage degradation, and also may contribute to an understanding of the bioactive peptides important in chondrocyte signaling.

Mammalian matrix metalloproteinase 28 (MMP-28) is expressed in several normal adult tissues, and during cutaneous wound healing. We show that, in frog and mouse embryos, MMP-28 is expressed predominantly throughout the nervous system. Xenopus expression increases during neurulation and remains elevated through early limb development where it is expressed in nerves. In the mouse, neural expression peaks at embryonic day (E) 14 but remains detectable through E17. During frog hindlimb regeneration XMMP-28 is not initially expressed in the regenerating nerves but is detectable before myelination. Following hindlimb denervation, XMMP-28 expression is detectable along regenerating nerves before myelination. In embryonic rat neuron-glial co-cultures, MMP-28 decreases after the initiation of myelination. Incubation of embryonic brain tissue with purified MMP-28 leads to the degradation of multiple myelin proteins. These results suggest that MMP-28 plays an evolutionarily conserved role in neural development and is likely to modulate the axonal-glial extracellular microenvironment., (2007 Wiley-Liss, Inc.)

Recent advances in the biological and analytical sciences have led to unprecedented interest in the discovery and quantitation of endogenous molecules that serve as indicators of drug safety, mechanism of action, efficacy, and disease state progression. By allowing for improved decision-making, these indicators, referred to as biomarkers, can dramatically improve the efficiency of drug discovery and development. Mass spectrometry has been a key part of biomarker discovery and evaluation owing to several important attributes, which include sensitive and selective detection, multi-analyte analysis, and the ability to provide structural information. Because of these capabilities, mass spectrometry has been widely deployed in search for new markers both through the analysis of large molecules (proteomics) and small molecules (metabonomics). In addition, mass spectrometry is increasingly being used to support quantitative measurement to assist in the evaluation and validation of biomarker leads. In this review, the dual role of mass spectrometry for biomarker discovery and measurement is explored for both large and small molecules by examining the key technologies and methods used along the continuum from drug discovery through clinical development.

Prostaglandin (PG) E(1) has been shown to possess anti-inflammatory properties and to modulate vascular reactivity. These activities are sometimes distinct from those of PGE(2), suggesting that endogenously produced PGE(1) may have some beneficial therapeutic effects compared with PGE(2). Increasing the endogenous formation of PGE(1) requires optimization of two separate processes, namely, enrichment of cellular lipids with dihomo-gamma-linolenic acid (20:3 n-6; DGLA) and effective cyclo-oxygenase-dependent oxygenation of substrate DGLA relative to arachidonic acid (AA; 20:4 n-6). DGLA and AA had similar affinities (K(m) values) and maximal reaction rates (V(max)) for cyclo-oxygenase-2 (COX-2), whereas AA was metabolized preferentially by cyclo-oxygenase-1 (COX-1). To overcome the kinetic preference of COX-1 for AA, CP-24879, a mixed Delta(5)/Delta(6) desaturase inhibitor, was used to enhance preferential accumulation of DGLA over AA in cells cultured in the presence of precursor gamma-linolenic acid (18:3 n-6). This protocol was tested in two cell lines and both yielded a DGLA/AA ratio of approx. 2.8 in the total cellular lipids. From the enzyme kinetic data, it was calculated that this ratio should offset the preference of COX-1 for AA over DGLA. PGE(1) synthesis in the DGLA-enriched cells was increased concurrent with a decline in PGE(2) formation. Nevertheless, PGE(1) synthesis was still substantially lower than that of PGE(2). It appears that employing a dietary or a combined dietary/pharmacological paradigm to augment the cellular ratio of DGLA/AA is not an effective route to enhance endogenous synthesis of PGE(1) over PGE(2), at least in cells/tissues where COX-1 predominates over COX-2.

A drastic increase of antifungal activity was demonstrated during plant seed germination and in seed protein extract in vitro. Multiple antifungal proteins with a wide spectrum of activity were generated and identified. Chromatographic and electrophoretic analysis demonstrated that during seed germination, more fractions with potent antifungal activity were generated, and the antifungal activity shifted from small molecules to high molecular proteins. This germination-related increase of antifungal activity were observed in all three plants tested, i.e., cheeseweed, cigar tree and wheat. This rapid increase of antifungal activity was also observed with incubation of seed proteins in vitro, suggesting that at least part of the antifungal protein generation is independent of gene expression. Seven antifungal proteins with activities against five different plant pathogens were isolated from the active fractions. However, random digestion of purified seed protein with multiple proteinases failed to generate any antifungal proteins. It is suggested that during plant seed germination, a regulated biochemical process takes place that results in the generation of multiple peptides or proteins with antifungal activities. This onset of antifungal proteins is transitional in nature, but could play an important role in the protection of plants in early stage of development when the more sophisticated defense system has yet to develop., ((C)2002 Elsevier Science (USA).)

Electrospray/tandem mass spectrometry was used to quantify lipid remodeling in mouse liver and plasma during inhibition of polyunsaturated fatty acid synthesis by the delta6 fatty acid desaturase inhibitor, SC-26196. SC-26196 caused increases in linoleic acid and corresponding decreases in arachidonic acid and docosahexaenoic acid in select molecular species of phosphatidylcholine, phosphatidylethanolamine, and cholesterol esters but not in phosphatidylserine, phosphatidylinositol, or triglycerides. For linoleic acid-, arachidonic acid-, and docosahexaenoic acid-containing phospholipid species, this difference was, in part, determined by the fatty acid at the sn-1 position, namely, palmitic or stearic acid. An understanding of phospholipid remodeling mediated by delta6 desaturase inhibition should aid in clarifying the contribution of arachidonic acid derived via de novo synthesis or obtained directly in the diet during inflammatory responses.

The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the alpha and beta chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4' positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases.

Decreased synthesis of arachidonic acid by inhibition of the Delta6 or Delta5 desaturase was evaluated as a means to mitigate inflammation. Using quantitative in vitro and in vivo radioassays, novel compounds representing five classes of Delta5 desaturase inhibitors and one class of Delta6 desaturase inhibitor were identified. The Delta6 desaturase inhibitor, SC-26196, had pharmacokinetic and pharmacodynamic profiles in mice that allowed for the evaluation of the pharmacological effects of chronic inhibition of desaturase activity. SC-26196 decreased edema to the same extent as indomethacin or essential fatty acid deficiency in the carrageenan paw edema model in the mouse. The antiinflammatory properties of SC-26196 were consistent with its mechanism of action as a Delta6 desaturase inhibitor: 1) A correlation existed between inhibition of liver Delta6 desaturase activity and decreases in edema. 2) The onset of the decrease in edema was time dependent. 3) Selective reduction of arachidonic acid occurred dose dependently in liver, plasma and peritoneal cells. 4) In the presence of SC-26196, controlled refeeding of arachidonic acid, but not oleic acid, reversed the changes resulting from desaturase inhibition. The Delta6 desaturase may be a target for development of antiinflammatory drugs whose mechanism of action is unique.