Efficacy and Safety of Dapagliflozin in Patients With CKD Across Major Geographic Regions.

Introduction: This study aimed to examine the efficacy and safety of dapagliflozin in the Dapagliflozin and Prevention of Adverse Outcomes in Chronic Kidney Disease (DAPA-CKD) trial (NCT03036150) by geographic region. Methods: Adults with chronic kidney disease (CKD) with or without type 2 diabetes, with estimated glomerular filtration rate (eGFR) 25 to 75 ml/min per 1.73 m2 and urinary albumin-to-creatinine ratio (UACR) 200 to 5000 mg/g were randomized to dapagliflozin (10 mg once daily) or placebo. The primary end point was a composite of a sustained decline in eGFR of ≥50%, end-stage kidney disease or death from kidney or cardiovascular causes. We categorized recruiting countries into 4 broad global regions: Asia, Europe, Latin America, and North America. Of 4304 randomized patients, 1346 (31.3%) were from Asia, 1233 (28.6%) from Europe, 912 (21.2%) from Latin America, and 813 (18.9%) from North America. Results: The relative risk of the primary composite end point was lower in patients randomized to dapagliflozin (relative to placebo) in all regions, with hazard ratios (95% CI) of 0.70 (0.48-1.00), 0.60 (0.43-0.85), 0.61 (0.43-0.86), and 0.51 (0.34-0.76) among patients from Asia, Europe, Latin America, and North America, respectively. There was no effect modification by region (interaction P = 0.77). Occurrence of serious adverse events (SAEs) was lower among patients randomized to dapagliflozin versus placebo (21.9% vs. 26.8%, 34.1% vs. 38.6%, 29.8% vs. 31.5%, and 34.9% vs. 41.0% in Asia, Europe, Latin America, and North America, respectively). Conclusion: Dapagliflozin reduced kidney and cardiovascular events and prolonged survival in patients with CKD, with and without type 2 diabetes, with no apparent effect modification by geographic region.

Roxadustat Versus Epoetin Alfa for Treating Anemia in Patients with Chronic Kidney Disease on Dialysis: Results from the Randomized Phase 3 ROCKIES Study.

BACKGROUND: Concerns regarding cardiovascular safety with current treatments for anemia in patients with dialysis-dependent (DD)-CKD have encouraged the development of alternatives. Roxadustat, an oral hypoxia-inducible factor prolyl hydroxylase inhibitor, stimulates erythropoiesis by increasing endogenous erythropoietin and iron availability. METHODS: In this open-label phase 3 study, patients with DD-CKD and anemia were randomized 1:1 to oral roxadustat three times weekly or parenteral epoetin alfa per local clinic practice. Initial roxadustat dose depended on erythropoiesis-stimulating agent dose at screening for patients already on them and was weight-based for those not on them. The primary efficacy end point was mean hemoglobin change from baseline averaged over weeks 28‒52 for roxadustat versus epoetin alfa, regardless of rescue therapy use, tested for noninferiority (margin, -0.75 g/dl). Adverse events (AEs) were assessed. RESULTS: Among 2133 patients randomized (n=1068 roxadustat, n=1065 epoetin alfa), mean age was 54.0 years, and 89.1% and 10.8% were on hemodialysis and peritoneal dialysis, respectively. Mean (95% confidence interval) hemoglobin change from baseline was 0.77 (0.69 to 0.85) g/dl with roxadustat and 0.68 (0.60 to 0.76) g/dl with epoetin alfa, demonstrating noninferiority (least squares mean difference [95% CI], 0.09 [0.01 to 0.18]; P<0.001). The proportion of patients experiencing ≥1 AE and ≥1 serious AE was 85.0% and 57.6% with roxadustat and 84.5% and 57.5% with epoetin alfa, respectively. CONCLUSIONS: Roxadustat effectively increased hemoglobin in patients with DD-CKD, with an AE profile comparable to epoetin alfa. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Safety and Efficacy Study of Roxadustat to Treat Anemia in Patients With Chronic Kidney Disease, on Dialysis. CLINICALTRIALS: gov Identifier: NCT02174731.

The dapagliflozin and prevention of adverse outcomes in chronic kidney disease (DAPA-CKD) trial: baseline characteristics.

BACKGROUND: The Dapagliflozin and Prevention of Adverse outcomes in Chronic Kidney Disease (DAPA-CKD; NCT03036150) trial was designed to assess the effect of the sodium-glucose co-transporter 2 (SGLT2) inhibitor dapagliflozin on kidney and cardiovascular events in participants with CKD with and without type 2 diabetes (T2D). This analysis reports the baseline characteristics of those recruited, comparing them with those enrolled in other trials. METHODS: In DAPA-CKD, 4304 participants with a urinary albumin:creatinine ratio (UACR) ≥200 mg/g and estimated glomerular filtration rate (eGFR) between 25 and 75 mL/min/1.73 m2 were randomized to dapagliflozin 10 mg once daily or placebo. Mean eGFR was 43.1 mL/min/1.73 m2 and median UACR was 949 mg/g (108 mg/mmol). RESULTS: Overall, 2906 participants (68%) had a diagnosis of T2D and of these, 396 had CKD ascribed to a cause other than diabetes. The most common causes of CKD after diabetes (n = 2510) were ischaemic/hypertensive nephropathy (n = 687) and chronic glomerulonephritis (n = 695), of which immunoglobulin A nephropathy (n = 270) was the most common. A total of 4174 participants (97%) were receiving an angiotensin-converting enzyme inhibitor or angiotensin receptor blocker, 1882 (43.7%) diuretics, 229 (5.3%) mineralocorticoid receptor antagonists and 122 (2.8%) glucagon-like peptide 1 receptor agonists. In contrast to the Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE), the DAPA-CKD trial enrolled participants with CKD due to diabetes and to causes other than diabetes. The mean eGFR of participants in the DAPA-CKD trial was 13.1 mL/min/1.73 m2 lower than in CREDENCE, similar to that in the Finerenone in Reducing Kidney Failure and Disease Progression in DKD (FIDELIO-DKD) trial and the Study Of diabetic Nephropathy with AtRasentan (SONAR). CONCLUSIONS: Participants with a wide range of underlying kidney diseases receiving renin-angiotensin system blocking therapy have been enrolled in the DAPA-CKD trial. The trial will examine the efficacy and safety of dapagliflozin in participants with CKD Stages 2-4 and increased albuminuria, with and without T2D.

Bioactive peptides and free amino acids profiles in different types of European dry-fermented sausages.

A wide variety of dry-fermented sausages are produced in European countries, where are considered valued traditional products. An intense proteolysis takes place during the processing of dry-fermented sausages due to the combined action of muscle and microbial peptidases, generating large amounts of peptides and free amino acids. These compounds participate in the development of the characteristic flavor of dry-fermented products, but some peptides can also exert certain bioactivities such as antioxidant and ACE inhibitory activities. This study has evaluated the changes in peptide profile and amino acid contents of three European dry-fermented sausages produced in Spain, Italy and Belgium, proving the intense degradation of proteins, mainly myofibrillar, and the generation of high amounts of different size peptides and free amino acids. The changes observed between the profiles of European sausages could be due to differences in product formulation, processing conditions and starter cultures used, which influence the activity of enzymes, both from muscle and bacterial origin. On the other hand, the bioactivity profile of each type of dry-fermented sausage was evaluated through the measurement of the ACE inhibitory and antioxidant activities in water-soluble peptide extracts fractionated by size-exclusion chromatography. Spanish and Belgian dry-fermented sausages showed values of ACE inhibition around 85%, whereas Belgian samples presented the highest DPPH radical-scavenging activity and ferric reducing power capacity. These results evidence the potential of Spanish, Italian and Belgian dry-fermented sausages as natural sources of bioactive peptides, giving an added-value to these traditional products.

Acetazolamide and N-acetylcysteine in the treatment of chronic mountain sickness (Monge's disease).

Patients suffering from chronic mountain sickness (CMS) have excessive erythrocytosis. Low -level cobalt toxicity as a likely contributor has been demonstrated in some subjects. We performed a randomized, placebo controlled clinical trial in Cerro de Pasco, Peru (4380m), where 84 participants with a hematocrit (HCT) ≥65% and CMS score>6, were assigned to four treatment groups of placebo, acetazolamide (ACZ, which stimulates respiration), N-acetylcysteine (NAC, an antioxidant that chelates cobalt) and combination of ACZ and NAC for 6 weeks. The primary outcome was change in hematocrit and secondary outcomes were changes in PaO2, PaCO2, CMS score, and serum and urine cobalt concentrations. The mean (±SD) hematocrit, CMS score and serum cobalt concentrations were 69±4%, 9.8±2.4 and 0.24±0.15µg/l, respectively for the 66 participants. The ACZ arm had a relative reduction in HCT of 6.6% vs. 2.7% (p=0.048) and the CMS score fell by 34.9% vs. 14.8% (p=0.014) compared to placebo, while the reduction in PaCO2 was 10.5% vs. an increase of 0.6% (p=0.003), with a relative increase in PaO2 of 13.6% vs. 3.0%. NAC reduced CMS score compared to placebo (relative reduction of 34.0% vs. 14.8%, p=0.017), while changes in other parameters failed to reach statistical significance. The combination of ACZ and NAC was no better than ACZ alone. No changes in serum and urine cobalt concentrations were seen within any treatment arms. ACZ reduced polycythemia and CMS score, while NAC improved CMS score without significantly lowering hematocrit. Only a small proportion of subjects had cobalt toxicity, which may relate to the closing of contaminated water sources and several other environmental protection measures.

Antilisterial peptides from Spanish dry-cured hams: Purification and identification.

The typical Spanish dry-cured ham has a particular sensory quality that makes it a distinctive food, highly appreciated for consumers worldwide. Its particular physicochemical properties, such as high salt content and reduced water activity contribute to their shelf-stability. However, post-processing actions carried out for the commercialization of these products such as slicing may increase the risk of development of pathogenic microorganisms as Listeria monocytogenes. During ripening, muscle proteins are hydrolyzed by muscle peptidases releasing peptides and free amino acids. Some of these peptides have been described to exert biological activities such as antioxidant and ACE-inhibition. In this study, a peptidomic strategy using mass spectrometry techniques has been used to identify and sequence those naturally generated peptides showing antilisterial activity. One hundred and five peptides have been identified in active fractions and some synthesized and their MIC calculated. Ten peptides were able to inhibit the growth of L. monocytogenes, being the pentapeptide RHGYM the most effective showing a MIC value of 6.25 mM. This study proves for the first time the potential antimicrobial action against L. monocytogenes of certain naturally generated peptides obtained from Spanish dry-cured ham.

Characterization of the peptide profile in Spanish Teruel, Italian Parma and Belgian dry-cured hams and its potential bioactivity.

Dry-cured hams are appreciated products in many European countries. One of the most important processes taking place during ham processing and responsible for its unique taste and flavour is the proteolysis of muscle proteins. Muscle peptidases play an important role in breaking down muscle proteins and generating small peptides and free amino acids. It is known that changes in genetics and processing conditions can result in differences in the action of endopeptidases and exopeptidases. In this study, the peptides generated in Spanish Teruel, Italian Parma and Belgian dry-cured hams have been identified and quantified using a label-free methodology to assess main differences in proteolysis between the 3 types of hams. The identification of the peptides resulted in differential peptide sequences according to the type of ham. On the other hand, an aqueous peptide extract fractionated by size-exclusion chromatography was assayed for Angiotensin-Converting Enzyme (ACE) inhibitory and antioxidant activity. Peptide fractions of Teruel ham exhibited 93% ACE inhibition while those from Parma and Belgian hams had ACE inhibitory activity of 70% and 76%, respectively. The investigated peptide fractions exhibited similar values of DPPH scavenging activity whereas an important Fe2+ reducing power was also detected in the same fractions, suggesting the important presence of peptides with antioxidant activity in the three studied types of dry-cured hams.

A peptidomic approach to study the contribution of added casein proteins to the peptide profile in Spanish dry-fermented sausages.

Peptidomics is a necessary alternative in the analysis of naturally generated peptides in dry-fermented processing. The intense proteolysis occurred during the processing of dry-fermented sausages is due to the action of endopeptidases and exopeptidases from both, endogenous muscle origin and lactic acid bacteria (LAB) added in the starter. Sodium caseinate is frequently used as an additive in this type of products because of its emulsifying properties, and consequently influences the protein profile available during the proteolysis. In this study, a mass spectrometry approach has been used to determine the impact of added sodium caseinate in the final peptide profile as well as to analyse its possible influence in the presence of certain previously described casein-derived bioactive peptides.

Small peptides hydrolysis in dry-cured meats.

Large amounts of different peptides are naturally generated in dry-cured meats as a consequence of the intense proteolysis mechanisms which take place during their processing. In fact, meat proteins are extensively hydrolysed by muscle endo-peptidases (mainly calpains and cathepsins) followed by exo-peptidases (mainly, tri- and di-peptidyl peptidases, dipeptidases, aminopeptidases and carboxypeptidases). The result is a large amount of released free amino acids and a pool of numerous peptides with different sequences and lengths, some of them with interesting sequences for bioactivity. This manuscript is presenting the proteomic identification of small peptides resulting from the hydrolysis of four target proteins (glyceraldehyde-3-phosphate dehydrogenase, beta-enolase, myozenin-1 and troponin T) and discusses the enzymatic routes for their generation during the dry-curing process. The results indicate that the hydrolysis of peptides follows similar exo-peptidase mechanisms. In the case of dry-fermented sausages, most of the observed hydrolysis is the result of the combined action of muscle and microbial exo-peptidases except for the hydrolysis of di- and tri-peptides, mostly due to microbial di- and tri-peptidases, and the release of amino acids at the C-terminal that appears to be mostly due to muscle carboxypeptidases.

Stability of ACE inhibitory ham peptides against heat treatment and in vitro digestion.

Angiotensin I-converting enzyme (ACE) inhibitory peptides derived from Spanish dry-cured ham have been examined for their stability during processing and after in vitro digestion. Results indicate that peptides preserved almost the same ACE inhibitory activity before and after applying diverse heating (from 50 to 117°C), times of processing (from 3 to 60min) and simulated in vitro digestion with gastrointestinal proteases. Peptides KAAAAP, AAPLAP, KPVAAP, IAGRP, and KAAAATP were the most potent peptides with IC50 values ranging from 12.37 to 25.94µM. Peptides IAGRP and PTPVP have also been identified in the processed sample (6min at 117°C), and in the in vitro digested sample. This study proves the high stability of ACE inhibitory peptides derived from Spanish dry-cured ham against temperature of processing and gastrointestinal digestion as well as the powerful ACE inhibitory activity of some of the peptides identified in Spanish dry-cured ham.

Identification of novel antioxidant peptides generated in Spanish dry-cured ham.

The objective of this study was to purify and identify antioxidant peptides present in a water soluble extract of Spanish dry-cured ham. The initial extract was loaded into a Sephadex G25 column and fractions showing antioxidant activity were collected, pooled together and subjected to reversed-phase chromatography for further purification. Using a nano-LC-MS/MS analysis, 27 peptides were identified in these fractions. Several key peptides were selected for synthesis and the determination of their antioxidant properties using the DPPH radical-scavenging assay and reducing power analysis. The strongest radical-scavenging activity was observed with peptide SAGNPN which showed 50% antioxidant activity at a concentration of 1.5mg/ml. On the other hand, the peptide GLAGA showed the higher reducing power with 0.5 units of absorbance at 700 nm at a concentration of 1mg/ml. Other synthesised sequences showed lower antioxidant activity. The results indicate the potential of Spanish dry-cured ham as a source of antioxidant peptides naturally generated during the dry-curing process.

Purification and Identification of antihypertensive peptides in Spanish dry-cured ham.

Novel sequences exhibiting in vitro ACE inhibitory activity as well as in vivo antihypertensive activity were identified from Spanish dry-cured ham. Water soluble peptide extracts from dry-cured ham were purified by size-exclusion chromatography and reversed-phase high performance liquid chromatography and then, further identification of sequences was carried out by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 73 peptide sequences were identified from active fractions presenting 100% homology with different Sus scrofa skeletal muscle proteins. All identified peptides showed Mr between 374 and 1610 and amino acid sequences between 5 and 14 amino acids in length. Considering the low molecular mass and structural requirements for ACE inhibition some of the identified peptides were synthesised and their IC(50) calculated. The most potent peptide was found to be AAATP (IC(50) value of 100 µM). This peptide also showed good in vivo activity because it decreased systolic blood pressure by -25.62 ± 4.5 mmHg (p<0.05) in spontaneous hypertensive rats after 8 h administration. Other sequences yielded a moderate ACE inhibition. Results from this study show that Spanish dry-cured ham may represent a source of natural peptides with potential benefit for human health.

Cardiovascular and renal effects of chronic exposure to high altitude.

Over 140 million people live at high altitude, defined as living at an altitude of 2400 m or more above sea level. Subjects living under these conditions are continuously living under hypoxic conditions and, depending on the population, various adaptations have developed. Interestingly, subjects living chronically at high altitude appear to have a decreased frequency of obesity, diabetes and coronary artery disease. However, these benefits on health are balanced by the frequent development of systemic and pulmonary hypertension. Recently, it has been recognized that subjects living at high altitude are at risk for developing high-altitude renal syndrome (HARS), which is a syndrome consisting of polycythemia, hyperuricemia, systemic hypertension and microalbuminuria, but with preserved glomerular filtration rate. More studies should be performed to characterize the mechanisms and etiology of HARS; as such studies may be of benefit not only to the high-altitude population, but also to better understanding of the renal consequences of acute and chronic hypoxia.

Antihypertensive effect and antioxidant activity of peptide fractions extracted from Spanish dry-cured ham.

This study examined the antihypertensive and antioxidant activities of water soluble fractions of a Spanish dry-cured ham extract. Antihypertensive activity of a fractionated peptide extract, by size-exclusion chromatography was determined by measuring changes in systolic blood pressure of spontaneously hypertensive rats after oral administration. Every sample exhibited antihypertensive activity, pooled fractions corresponding to 1700 Da or lower were the most antihypertensive with a decrease of 38.38 mm Hg in systolic blood pressure. In vitro experiments revealed marked in vitro angiotensin I-converting enzyme inhibitory activity in fractions corresponding to these elution volumes. Some of the fractions exhibited great 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity, ranging from 39% to 92% as well as superoxide ion extinguishing ability with values ranging from 41.67% to 50.27% of the antioxidant activity, suggesting the presence of peptides with antioxidant activity. These findings suggest that Spanish dry-cured ham contains peptides with antioxidant and antihypertensive activities.

Antihypertensive activity of peptides identified in the in vitro gastrointestinal digest of pork meat.

This study investigated the in vivo antihypertensive activity of three novel peptides identified in the in vitro digest of pork meat. These peptides were RPR, KAPVA and PTPVP and all of them showed significant antihypertensive activity after oral administration to spontaneously hypertensive rats, RPR being the peptide with the greatest in vivo activity. To our knowledge, this is the first report showing the in vivo antihypertensive action of the three peptides from nebulin (RPR) and titin (KAPVA and PTPVP), thus confirming their reported in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. These findings suggest that pork meat could constitute a source of bioactive constituents that could be utilized in functional foods or nutraceuticals.

Possible biological markers of the time of processing of dry-cured ham.

Several muscle compounds (creatine, creatinine, hypoxanthine, inosine, inosine 5' monophosphate, xanthine, adenosine monophosphate, guanosine, and uridine) were studied as possible biological markers of a minimum dry-cured ham processing time. A correlation between the concentration of the compounds and the time of processing was found. The ratios for some of them were calculated to study their behaviour during processing. The Hx/Ino ratio substantially increased up to 5 months of curing and then remained constant (p<0.05). The Hx/Ino ratio might be considered as a potential biomarker of the minimum time of dry-cured processing (5 months). The Cn/Cr ratio increased during drying up to 9 months of ripening (p<0.05). However, although Cn/Cr ratios remained constant after 9 months of processing, variations between hams were observed due to the differences existing in the raw meats and small differences in processing conditions, making it difficult to consider Cn/Cr ratios as biomarkers of ripening time.

High altitude renal syndrome (HARS).

More than 140 million people live permanently at high altitude (>2400 m) under hypoxic conditions that challenge basic physiology. Here we present a short historical review of the populating of these regions and of evidence for genetic adaptations and environmental factors (such as exposure to cobalt) that may influence the phenotypic responses. We also review some of the common renal physiologic responses focusing on clinical manifestations. The frequent presentation of systemic hypertension and microalbuminuria with relatively preserved GFR coupled with the presence of polycythemia and hyperuricemia suggests a new clinical syndrome we term high altitude renal syndrome (HARS). ACE inhibitors appear effective at reducing proteinuria and lowering hemoglobin levels in these patients.

Characterization of peptides released by in vitro digestion of pork meat.

The main objective of this work was to identify and characterize the peptides generated by simulated gastrointestinal digestion of pork meat (longissimus dorsi) by the sequential action of pepsin and pancreatin. The obtained hydrolysate was analyzed by liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer equipped with a nanoelectrospray ionization source (nano LC-ESI-MS/MS). Using this technique 51 different peptides were identified in the hydrolysate, corresponding to fragments of the main structural muscle proteins and some well-known sarcoplasmic proteins. To the best of our knowledge, this constitutes the highest number of peptides identified in pork meat digests. Peptide fragment size ranged from six to sixteen amino acids, being rich in proline residues and thus making them more resistant to further degradation by digestive enzymes. The present study constitutes a clear evidence of the extensive degradation that pork muscle proteins would undergo after gastrointestinal digestion, giving rise to a wide variety of short peptides. So, the use of in vitro digestion contributes to a better knowledge about the generation of peptides from diets with high protein quality.