KEY POINTS:Lower urinary EGF levels, normalized to urinary creatinine, reflected impaired tubular health and were associated with higher kidney risk across CKD. Sodium-glucose cotransporter 2 inhibitors attenuated the decline in urinary EGF-creatinine ratio observed with placebo, supporting a role in maintaining tubular health. Early increases in urinary EGF-creatinine ratio were linked to lower kidney risk, adding prognostic value beyond albuminuria. BACKGROUND:Urinary EGF is a marker of tubular repair capacity. Lower urinary EGF-creatinine ratio (uEGF/Cr) levels associate with kidney disease progression in patients with type 2 diabetes at early stages of CKD. In these patients, sodium-glucose cotransporter 2 inhibitors (SGLT2is) are associated with increased tubular EGF expression. In this study, we aimed to extend these findings to a broad CKD population with and without type 2 diabetes at various stages of CKD. METHODS:We measured EGF in stored urine samples at baseline and year 1 in participants from the Canagliflozin Cardiovascular Assessment Study (CANVAS), Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE), and Dapagliflozin and Prevention of Adverse Outcomes in CKD (DAPA-CKD) clinical trials. Associations of baseline and longitudinal uEGF/Cr with the composite kidney outcome (sustained ≥40% eGFR decline, kidney failure, or kidney-related death) were assessed using multivariable Cox regression, and associations with annual eGFR decline using a two-slope linear mixed-effects model. Treatment effects of SGLT2i on uEGF/Cr over time were analyzed with analysis of covariance. RESULTS:In participants with type 2 diabetes from the CANVAS and CREDENCE trials ( N =5978), higher baseline uEGF/Cr was associated with a lower risk of the composite kidney outcome (hazard ratio per two-fold higher uEGF/Cr, 0.87 [95% confidence interval, 0.80 to 0.94]). SGLT2i attenuated the decline in uEGF/Cr over 1 year compared with placebo by 6.7% (95% confidence interval, 2.5 to 10.8). Increases in uEGF/Cr from baseline to year 1 were independently associated with a lower risk of the kidney outcome, even after accounting for 1-year changes in albuminuria, eGFR, and other clinical variables. Replication analyses showed similar results in the DAPA-CKD trial ( N =2450), with consistent findings in participants with and without diabetes. CONCLUSIONS:These results extend previous findings, supporting uEGF/Cr as a robust, independent biomarker of tubular health and kidney risk across diverse CKD populations. SGLT2i attenuated uEGF/Cr decline, and early changes in uEGF/Cr provided prognostic information beyond albuminuria.

PTEN hamartoma tumour syndrome (PHTS) is an inherited syndrome caused by germline loss of function of the phosphatase and tensin homologue (PTEN) tumour suppressor gene. This causes cancer predisposition and a range of other features including vascular anomalies and neurodevelopmental disorders, notably autism spectrum disorder (ASD). Emerging evidence indicates that PTEN is a central regulator of metabolism, and its loss produces profound changes in insulin– phosphatidylinositol 3-kinase (PI3K)–Akt–mechanistic target of rapamycin (mTOR) signalling, affecting glucose homeostasis, adiposity and energy balance. Paradoxically, individuals with PHTS may exhibit enhanced insulin sensitivity despite increased risk of obesity. The metabolic features of PHTS are not yet fully characterised but potentially influence cancer risk, development and progression of benign tumours and vascular anomalies, and ASD. Progressive weight gain is likely to increase the risk of heart failure, lower limb joint failure and sleep apnoea, and to exacerbate impaired mobility due to PHTS-related overgrowth or vascular anomalies. This review explores the role of PTEN in metabolic regulation and evaluates the potential use of established medicines for type 2 diabetes and obesity to treat cardiometabolic and other features of PHTS. Metabolic interventions are an unexplored potential therapeutic avenue that could be important for people with PHTS.

Endothelin-1 (ET-1) was discovered in 1988, followed by identification of ETA and ETB receptors in 1990, enabling rapid development of the first endothelin receptor antagonists (bosentan, ambrisentan, and macitentan) for pulmonary arterial hypertension, a condition marked by elevated ET-1. Nearly a decade later, a new therapeutic wave began with the ETB agonist sovateltide (2021) for cerebral ischemic stroke, demonstrating the benefits of ETB activation. More recent antagonist development has shifted toward ETA-selective agents to preserve ETB function. Clazosentan (ETA) for cerebral vasospasm and aprocitentan (ETA/ETB) for resistant hypertension extended endothelin-targeted therapy into more common diseases. In kidney disease, sparsentan (AT1/ETA) and atrasentan (ETA) have both been approved for IgA nephropathy, with atrasentan succeeding after earlier failed trials. Repurposing has become a major strategy in G protein-coupled receptor drug development. This review outlines approaches for identifying legacy endothelin compounds suitable for new indications, using zibotentan, now combined with dapagliflozin to reduce fluid retention as an example. Kidney disease remains a central focus of current trials, which include new ETA-selective diosuxentan and monoclonal antibody getagozumab, and the ETB peptide antagonist vodudeutentan. The review summarizes recent pharmacology and clinical data and highlights emerging strategies for next-generation endothelin-pathway therapeutics.

INTRODUCTION:Dapagliflozin improves kidney outcomes in patients with chronic kidney disease (CKD), yet the underlying mechanisms by which it exerts protective effects are not fully elucidated. Here, we applied plasma proteomics to identify proteins and pathways linked to CKD progression and examined which of these are modulated by dapagliflozin. METHODS:We measured 3072 circulating plasma proteins (Olink, Uppsala, Sweden) in 2485 of the 4304 (57.7%) patients with CKD from the DAPA-CKD trial (eGFR 25-75 mL/min per 1.73 m2 and urinary albumin-to-creatinine ratio 200-5000 mg/g). Proteins associated with the composite kidney outcome (50% eGFR decline, kidney failure, and kidney death) and with dapagliflozin treatment were respectively identified by multivariable Cox proportional hazard regression and ANCOVA. Ingenuity pathway analysis was used to identify relevant molecular pathways and upstream regulators. RESULTS:Pathways that had the strongest association with the composite kidney outcome were hepatic fibrosis, insulin-like growth factor transport regulation, and tumor necrosis factor signaling. These pathways were generally related to fibrosis and inflammation. After 12 months, dapagliflozin treatment showed a (placebo-corrected) modification of 216 proteins, of which kidney injury molecule-1 showed the strongest reduction (-14.9%; 95% confidence interval: -17.9, -11.7). Of the 137 pathways associated with the kidney outcome, 35 (25%) were modified by dapagliflozin. Among these, activity of 11 (31% of 35) pathways decreased, and 10 (29% of 35) pathways were increased. Pathways associated with kidney outcome and modulated by dapagliflozin were related to extracellular matrix remodeling, inflammation and fibrosis, and immune-vascular interactions. CONCLUSIONS:This proteomic analysis reveals mechanisms underlying dapagliflozin's beneficial effects in CKD progression and shows that key pathways associated with CKD progression are modified by dapagliflozin, supporting its anti-inflammatory and antifibrotic potential.

BACKGROUND:We investigated circulating protein profiles and molecular pathways among various chronic kidney disease (CKD) aetiologies to study its underlying molecular heterogeneity. METHODS:We conducted a proteomic biomarker analysis in the DAPA-CKD trial recruiting adults with and without type 2 diabetes with an estimated glomerular filtration rate of 25-75 ml/min/1.73 m2 and a urine albumin:creatinine ratio of 200-5000 mg/g. A total of 2926 proteins were analysed by Olink Explore in plasma samples collected at baseline. Proteins associated with diabetic kidney disease (DKD), glomerulonephritis (GN) and hypertensive nephropathy were identified using linear models to compare each CKD aetiology against the others. RESULTS:Of 4304 randomized participants, baseline plasma proteomic profiles were available for 2485 participants (57.7%). Compared with patients without DKD, those with DKD had higher plexin B2 (PLXNB2; log2 fold difference 0.34, P < .001), vascular adhesion protein-1 (VAP-1; log2 fold difference 0.34, P < .001) and kidney injury molecule-1 (KIM-1; log2 fold difference 0.79, P < .001), suggesting involvement of immune activation, oxidative stress and tubular damage. Based on these molecular pathways, interleukin-6 was identified as an upstream regulator in DKD. In an external database of the Kidney Precision Medicine Project, PLXNB2 and VAP-1 were also elevated in DKD compared with non-DKD (log2 fold difference 0.41 and 0.42, respectively). In GN, levels of nephrin, a key slit diaphragm protein, were lower (log2 fold difference -0.27, P < .001). Furthermore, receptor for advanced glycation end products (log2 fold difference 0.28, P < .001) and heparan sulphate 6-O-sulfotransferase 2 (log2 fold difference 0.2, P < .05) were higher, suggesting glycocalyx disruption and inflammation. In hypertensive nephropathy, epiregulin, a member of the epidermal growth factor family, was higher compared with other CKD aetiologies. CONCLUSION:Our study highlights distinct proteomic differences in patients with DKD, GN and hypertensive nephropathy. These findings may aid in unravelling the heterogeneous underlying molecular phenotype of CKD pathophysiology.

AIMS:To evaluate whether type 2 diabetes status modifies the efficacy and safety of combining zibotentan (zibo), a selective endothelin receptor antagonist, and dapagliflozin (dapa) compared to placebo plus dapagliflozin in individuals with chronic kidney disease (CKD). METHODS AND MATERIALS:We conducted a post hoc analysis of the ZENITH-CKD trial, a multicentre 12-week, double-blind, randomized, active-controlled, phase 2b study involving 447 participants with CKD (261 with and 186 without type 2 diabetes). Participants were assigned to zibotentan (0.25 or 1.5 mg) plus dapagliflozin 10 mg or placebo plus dapagliflozin 10 mg. Changes in urinary albumin-to-creatinine ratio (UACR) and markers of fluid retention (bodyweight and B-type natriuretic peptide [BNP]) were compared in participants with and without type 2 diabetes. RESULTS:Zibo/dapa 0.25/10 mg changed UACR by -37.7% (90% CI: -40.4, -23.4) compared to placebo/dapa in participants without diabetes and by -17.9% (90% CI: -31.3, -2.0) in participants with diabetes (p-interaction 0.096). Effects of zibo/dapa 1.5/10 mg on UACR were consistent regardless of diabetes status (-34.0% (90% CI: -45.0, -20.8) vs. -33.0% (90% CI: -42.2, -22.5), p-interaction 0.921). Changes in body weight and BNP did not differ by diabetes status. Fluid retention occurred in five participants with diabetes assigned to zibo/dapa 1.5/10 mg and one participant with diabetes in the zibo/dapa 0.25/10 mg group. Fluid retention did not occur in those without diabetes in both zibo/dapa groups. With placebo/dapa, fluid retention occurred in one participant without diabetes and in none with diabetes. CONCLUSIONS:Combination therapy with zibotentan and dapagliflozin demonstrated consistent efficacy and safety across CKD patients with and without type 2 diabetes.

Nitrogen-driven water conservation during states of environmental energy and water scarcity is part of an evolutionary conserved physiological principle in many vertebrate and invertebrate organisms, known as aestivation. Because evolutionary reconstruction suggests that early Homo species evolved in habitats with constant access to water, this ancient biological axiom is considered redundant in human physiology. We have recently shown that therapeutic sodium/glucose cotransporter 2 inhibition (SGLT2i) in patients with chronic heart failure promotes urinary glucose excretion, but does not have an osmotic-diuretic or natriuretic effect [1]. It is therefore unclear how SGLT2i may chronically decongest patients with heart failure without increasing renal water excretion. We tested the hypothesis that SGLT2i triggers evolutionary conserved, aestivation-like metabolic patterns for water conservation in humans by activating nitrogen/urea-driven water conservation, with increased ureagenesis and reduced fractional urea excretion. DAPA-Shuttle1 was an investigator-initiated, mechanistic, randomized, placebo-controlled clinical trial designed to compare the effects of dapagliflozin 10 mg versus matching placebo on renal water and electrolyte handling and water conservation metabolism after 48 h (early effect) and after 4 weeks (late effect) of treatment in patients with heart failure NYHA classes I and II. 29 participants (placebo: n =14; dapagliflozin: n =15) completed the study with accurate 24 h urine collections (mean age 59 ± 14 years, 48.3% with type 2 diabetes). SGLT2i increased free amino acids, their derived ketoacids, and ornithine/arginine levels in the 24 h urine metabolome, suggesting increased urea production. Instead of increasing urea excretion in response to promoted ureagenesis, SGLT2i reduced FEUrea by −6% (p < 0.01), which resulted in increased urine concentration (p < 0.01) and elevated blood urea levels (p < 0.05). This amino acid-dependent modulation of water metabolism included increased generation of specific short-chain ketoacids that reduce mitochondrial metabolic rate and water production. As a result, SGLT2i established a new steady-state in water homeostasis with mild under-hydration and nitrogen-driven water conservation, which abolished the osmotic-diuretic potential of glucosuria within 24–48 hours of treatment initiation. Although they significantly increase glucosuria, SGLT2i do not have an osmotic diuretic effect—instead, they increase urine concentration through a reduction in fractional urea excretion and parallel promotion of ureagenesis. The resulting metabolic switches for urea production increase plasma urea levels and favour energy-efficient ketone body fuel production. The reduction in mitochondrial metabolic rate results in a new steady-state of body water homeostasis with mild dehydration, which may explain decongestion even in the absence of a diuretic effect. These metabolic features, often considered as explanations for the beneficial effects of SGLT2i on healthspan and longevity, closely resemble the metabolic adaptations seen in aestivating organisms. Trial registration: https://clinicaltrials.gov; unique identifier NCT04080518. Funding: AstraZeneca externally sponsored research program (ESR-18-13712), German Ministry for Economic Affairs and Climate Action (50WB0920, 50WB1624, 50WB2024, and 50WB2324).

Cannabinoid receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Cannabinoid Receptors [127]) are activated by endogenous ligands that include N-arachidonoylethanolamine (anandamide), N-homo-γ-linolenoylethanolamine, N-docosatetra-7,10,13,16-enoylethanolamine and 2-arachidonoylglycerol. Potency determinations of endogenous agonists at these receptors are complicated by the possibility of differential susceptibility of endogenous ligands to enzymatic conversion [5].There are currently three licenced cannabinoid medicines each of which contains a compound that can activate CB1 and CB2 receptors [119]. Two of these medicines were developed to suppress nausea and vomiting produced by chemotherapy. These are nabilone (Cesamet®), a synthetic CB1/CB2 receptor agonist, and synthetic Δ9-tetrahydrocannabinol (Marinol®; dronabinol), which can also be used as an appetite stimulant. The third medicine, Sativex®, contains mainly Δ9-tetrahydrocannabinol and cannabidiol, both extracted from cannabis, and is used to treat multiple sclerosis and cancer pain.

AIMS:We assessed the effects of dapagliflozin on natriuresis and blood pressure in patients with type 2 diabetes and chronic kidney disease (CKD) and evaluated the consistency of these effects across patients with type 2 diabetes without CKD and CKD without type 2 diabetes. MATERIALS AND METHODS:We conducted a mechanistic, nonrandomised, open-label study to evaluate the effects of dapagliflozin on sodium excretion and blood pressure in patients with type 2 diabetes and CKD under a standardised sodium intake (150 mmol/day). Evaluations were performed at baseline (average of Days -3 to -1), treatment start (average of Days 2-4; hereafter referred to as Day 4), treatment end (average of Days 12-14; hereafter referred to as Day 14) and washout (average of Days 15-17). Similar protocols were applied to the type 2 diabetes without CKD and CKD without type 2 diabetes strata. Data from all strata were pooled to assess the consistency of the effects of dapagliflozin. RESULTS:In 13 participants with type 2 diabetes and CKD, 24-h sodium excretion did not increase during treatment, as reflected by changes from baseline (Day 4 change: -0.5 mmol/24-h [95% CI -15.4, 14.5], p = 0.95; Day 14 change: -11.7 mmol/24-h [95% CI -30.4, 7.1]). Compared to baseline, 24-h systolic blood pressure decreased by 6.0 mmHg [95% CI -19.8, 7.7] at Day 4 and by 3.7 mmHg [95% CI -13.7, 6.2] at Day 14. In the pooled stratum of 33 participants, 24-h sodium excretion remained unchanged relative to baseline (Day 4 change: -7.4 mmol/24-h [95% CI -16.2, 1.5], p = 0.10; Day 14 change: -10.1 mmol/24-h [95% CI -25.1, 5.0], p = 0.18), whereas 24-h systolic blood pressure was consistently reduced by 6.8 mmHg ([95% CI -10.4, -3.3], p < 0.001) at Day 4 and by 6.5 mmHg ([95% CI -10.1, -3.0], p < 0.001) at Day 14, with no evidence of heterogeneity across strata. CONCLUSIONS:Dapagliflozin reduced blood pressure during standardised sodium intake in patients with type 2 diabetes and/or CKD without increasing sodium excretion, suggesting that the blood pressure-lowering effects occur through mechanisms beyond natriuresis.

Previous cross-sectional transcriptomics studies on diabetic kidney disease (DKD) kidney tissue have shown correlations between gene expression and both disease status and kidney function at the time of biopsy; however, longitudinal data are scarce. We utilized clinical follow-up data up to five years post-biopsy, linking the transcriptomes of diagnostic kidney biopsies to progression rates and outcomes in 19 patients with DKD. Patients were stratified into “rapid progressors” and “non-rapid progressors” based on clinical parameters (eGFR slope, CKD stage advancement, degree of albuminuria, composite outcome of kidney failure or 40

Roux-en-Y gastric bypass (GBP) surgery is an effective treatment for reducing body weight and correcting metabolic dysfunction in individuals with severe obesity. Herein, we characterize the differences between very low energy diet (VLED) and GBP induced weight loss by multi-omic analyses of microbiome and host features in a non-randomized, controlled, single-center study. Eighty-eight participants with severe obesity were recruited into two arms - GBP versus VLED with matching weight loss for 6 weeks and 2-years of follow-up. A dramatic shift in the distribution of gut microbial taxa and their functional capacity was seen in the GBP group at Week 2 after surgery and was sustained through 2 years. Multi-omic analyses were performed after 6 weeks of matching weight loss between the GBP and VLED groups, which pointed to microbiome derived metabolites such as indoxyl sulphate as characterizing the GBP group. We also identified an inverse association between Streptococcus parasanguinis (an oral commensal) and plasma levels of tryptophan and tyrosine. These data have important implications, as they reveal a significant robust restructuring of the microbiome away from a baseline dysbiotic state in the GBP group. Furthermore, multi-omics modelling points to potentially novel mechanistic insights at the intersection of the microbiome and host.

IntroductionPrimary sclerosing cholangitis (PSC) is a chronic liver disease of unknown cause contributing to cirrhosis and cancer but has no cure. PSC is characterized by inflammation within ductal fibrosis, progressive bile duct narrowing and loss, with damage to cholangiocytes (epithelial cells affecting bile production) and liver repair. ET-1, produced by cholangiocytes, contributes to fibrosis, vasoconstriction, and inflammation via ETA receptors. In patients, ET-1 and ETA gene expression are elevated and ETA antagonists reduce disease progression in PSC animal models. Ongoing clinical trials of portal hypertension in liver disease are testing the efficacy of a new treatment strategy combining ETA-selective antagonist zibotentan with SGLT2 inhibitor dapagliflozin.MethodsTo interrogate the potential of a comparable strategy in PSC we have initially compared the localization of ET receptors and SGLT2 transporter in human PSC liver.ResultsIn ethically sourced healthy human liver, ETA immunofluorescence was primarily found in bile duct epithelial cells within the portal tract, smooth muscle of the central vein, with low levels in hepatocytes. SGLT2 immunofluorescence was mainly detected on bile duct epithelial cells and hepatocytes. ETA co-localized with smooth muscle cells in large arteries and veins, while ETB immunoreactivity was present in hepatocytes and endothelial cells. In the PSC vasculature, the pattern of expression of smooth muscle ETA receptors that mediate vasoconstriction was retained, consistent with the hypothesis that ETA selective antagonists would be beneficial in reducing portal hypertension. ETB receptors were principally localised on endothelial cells and would be expected to mediate beneficial vasodilation. In diseased areas, all three proteins localised to ductal reactions, reflecting the response of the liver to injury, involving cholangiocyte proliferation, promoting beneficial regeneration but also associated with fibrosis and inflammation. Both ETA, ETB and low levels of SGLT2 immunofluorescence localised to fibroblasts within the fibrous septa where bands of scar tissue can restrict hepatic blood flow, leading to cirrhosis.DiscussionBoth drug targets were retained in the key hallmarks of PSC pathology; ETA and SGLT2 staining within cholangiocytes undergoing ductal transformation and cells within the fibrotic septa, supporting the proposed benefit of combination treatment strategy.