Renal Mitochondrial ATP Transporter Ablation Ameliorates Obesity-Induced CKD.

SIGNIFICANCE STATEMENT: This study sheds light on the central role of adenine nucleotide translocase 2 (ANT2) in the pathogenesis of obesity-induced CKD. Our data demonstrate that ANT2 depletion in renal proximal tubule cells (RPTCs) leads to a shift in their primary metabolic program from fatty acid oxidation to aerobic glycolysis, resulting in mitochondrial protection, cellular survival, and preservation of renal function. These findings provide new insights into the underlying mechanisms of obesity-induced CKD and have the potential to be translated toward the development of targeted therapeutic strategies for this debilitating condition. BACKGROUND: The impairment in ATP production and transport in RPTCs has been linked to the pathogenesis of obesity-induced CKD. This condition is characterized by kidney dysfunction, inflammation, lipotoxicity, and fibrosis. In this study, we investigated the role of ANT2, which serves as the primary regulator of cellular ATP content in RPTCs, in the development of obesity-induced CKD. METHODS: We generated RPTC-specific ANT2 knockout ( RPTC-ANT2-/- ) mice, which were then subjected to a 24-week high-fat diet-feeding regimen. We conducted comprehensive assessment of renal morphology, function, and metabolic alterations of these mice. In addition, we used large-scale transcriptomics, proteomics, and metabolomics analyses to gain insights into the role of ANT2 in regulating mitochondrial function, RPTC physiology, and overall renal health. RESULTS: Our findings revealed that obese RPTC-ANT2-/- mice displayed preserved renal morphology and function, along with a notable absence of kidney lipotoxicity and fibrosis. The depletion of Ant2 in RPTCs led to a fundamental rewiring of their primary metabolic program. Specifically, these cells shifted from oxidizing fatty acids as their primary energy source to favoring aerobic glycolysis, a phenomenon mediated by the testis-selective Ant4. CONCLUSIONS: We propose a significant role for RPTC-Ant2 in the development of obesity-induced CKD. The nullification of RPTC-Ant2 triggers a cascade of cellular mechanisms, including mitochondrial protection, enhanced RPTC survival, and ultimately the preservation of kidney function. These findings shed new light on the complex metabolic pathways contributing to CKD development and suggest potential therapeutic targets for this condition.

Renal Endocannabinoid Dysregulation in Obesity-Induced Chronic Kidney Disease in Humans.

The endocannabinoid system (ECS) regulates various physiological processes, including energy homeostasis and kidney function. ECS upregulation in obese animals and humans suggests a potential link to obesity-induced chronic kidney disease (CKD). However, obesity-induced ECS changes in the kidney are mainly studied in rodents, leaving the impact on obese humans unknown. In this study, a total of 21 lean and obese males (38-71 years) underwent a kidney biopsy. Biochemical analysis, histology, and endocannabinoid (eCB) assessment were performed on kidney tissue and blood samples. Correlations between different parameters were evaluated using a comprehensive matrix. The obese group exhibited kidney damage, reflected in morphological changes, and elevated kidney injury and fibrotic markers. While serum eCB levels were similar between the lean and obese groups, kidney eCB analysis revealed higher anandamide in obese patients. Obese individuals also exhibited reduced expression of cannabinoid-1 receptor (CB1R) in the kidney, along with increased activity of eCB synthesizing and degrading enzymes. Correlation analysis highlighted connections between renal eCBs, kidney injury markers, obesity, and related pathologies. In summary, this study investigates obesity's impact on renal eCB "tone" in humans, providing insights into the ECS's role in obesity-induced CKD. Our findings enhance the understanding of the intricate interplay among obesity, the ECS, and kidney function.

Generation and characterization of a mouse model for one anastomosis gastric bypass surgery.

One anastomosis gastric bypass (OAGB) surgery became a common bariatric procedure in recent years. In this surgery, the distal stomach, duodenum, and proximal jejunum are bypassed, leading to weight loss, improvement in metabolic parameters, and a change in hormonal secretion. We sought to generate and characterize a mouse model for OAGB. Mice fed for 26 wk on a high-fat diet were assigned to OAGB, sham surgery, or caloric restriction and were followed for 50 more days on a high-fat diet. Physiological and histological parameters of the mice were compared during and at the end of the experiment. OAGB-operated mice lost weight and displayed low levels of plasma lipids, high insulin sensitivity, and rapid glucose metabolism compared with sham-operated mice. OAGB-operated mice had higher energy expenditure, higher levels of glucagon-like peptide (GLP-1), and lower albumin than weight-matched calorie-restricted mice. There was no difference in the histology of the endocrine pancreas. The livers of OAGB mice had little hepatic steatosis yet presented with a large number of phagocytic cells. The OAGB mouse model recapitulates many of the phenotypes described in patients that underwent OAGB and enables molecular and physiological studies on the outcome of this surgery.NEW & NOTEWORTHY A mouse model for one anastomosis gastric bypass (OAGB) surgery displays similar outcomes to clinical reports and enables to study the weight loss-dependent and -independent effects of this bariatric surgery.

The Metabolic Efficacy of a Cannabidiolic Acid (CBDA) Derivative in Treating Diet- and Genetic-Induced Obesity.

Obesity is a global medical problem; its common form is known as diet-induced obesity (DIO); however, there are several rare genetic disorders, such as Prader-Willi syndrome (PWS), that are also associated with obesity (genetic-induced obesity, GIO). The currently available therapeutics for treating DIO and GIO are very limited, and they result in only a partial improvement. Cannabidiolic acid (CBDA), a constituent of Cannabis sativa, gradually decarboxylates to cannabidiol (CBD). Whereas the anti-obesity properties of CBD have been reasonably identified, our knowledge of the pharmacology of CBDA is more limited due to its instability. To stabilize CBDA, a new derivative, CBDA-O-methyl ester (HU-580, EPM301), was synthesized. The therapeutic potential of EPM301 in appetite reduction, weight loss, and metabolic improvements in DIO and GIO was tested in vivo. EPM301 (40 mg/kg/d, i.p.) successfully resulted in weight loss, increased ambulation, as well as improved glycemic and lipid profiles in DIO mice. Additionally, EPM301 ameliorated DIO-induced hepatic dysfunction and steatosis. Importantly, EPM301 (20 and 40 mg/kg/d, i.p.) effectively reduced body weight and hyperphagia in a high-fat diet-fed Magel2null mouse model for PWS. In addition, when given to standard-diet-fed Magel2null mice as a preventive treatment, EPM301 completely inhibited weight gain and adiposity. Lastly, EPM301 increased the oxidation of different nutrients in each strain. All together, EPM301 ameliorated obesity and its metabolic abnormalities in both DIO and GIO. These results support the idea to further promote this synthetic CBDA derivative toward clinical evaluation in humans.

Agonist of RORA Attenuates Nonalcoholic Fatty Liver Progression in Mice via Up-regulation of MicroRNA 122.

BACKGROUND & AIMS: Development of nonalcoholic steatohepatitis (NASH) is associated with reductions in hepatic microRNA122 (MIR122); the RAR related orphan receptor A (RORA) promotes expression of MIR122. Increasing expression of RORA in livers of mice increases expression of MIR122 and reduces lipotoxicity. We investigated the effects of a RORA agonist in mouse models of NASH. METHODS: We screened a chemical library to identify agonists of RORA and tested their effects on a human hepatocellular carcinoma cell line (Huh7). C57BL/6 mice were fed a chow or high-fat diet (HFD) for 4 weeks to induce fatty liver. Mice were given hydrodynamic tail vein injections of a MIR122 antagonist (antagomiR-122) or a control antagomiR once each week for 3 weeks while still on the HFD or chow diet, or intraperitoneal injections of the RORA agonist RS-2982 or vehicle, twice each week for 3 weeks. Livers, gonad white adipose, and skeletal muscle were collected and analyzed by reverse-transcription polymerase chain reaction, histology, and immunohistochemistry. A separate group of mice were fed an atherogenic diet, with or without injections of RS-2982 for 3 weeks; livers were analyzed by immunohistochemistry, and plasma was analyzed for levels of aminotransferases. We analyzed data from liver tissues from patients with NASH included in the RNA-sequencing databases GSE33814 and GSE89632. RESULTS: Injection of mice with antagomiR-122 significantly reduced levels of MIR122 in plasma, liver, and white adipose tissue; in mice on an HFD, antagomiR-122 injections increased fat droplets and total triglyceride content in liver and reduced ß-oxidation and energy expenditure, resulting in significantly more weight gain than in mice given the control microRNA. We identified RS-2982 as an agonist of RORA and found it to increase expression of MIR122 promoter activity in Huh7 cells. In mice fed an HFD or atherogenic diet, injections of RS-2982 increased hepatic levels of MIR122 precursors and reduced hepatic synthesis of triglycerides by reducing expression of biosynthesis enzymes. In these mice, RS-2982 significantly reduced hepatic lipotoxicity, reduced liver fibrosis, increased insulin resistance, and reduced body weight compared with mice injected with vehicle. Patients who underwent cardiovascular surgery had increased levels of plasma MIR122 compared to its levels before surgery; increased expression of plasma MIR122 was associated with increased levels of plasma free fatty acids and levels of RORA. CONCLUSIONS: We identified the compound RS-2982 as an agonist of RORA that increases expression of MIR122 in cell lines and livers of mice. Mice fed an HFD or atherogenic diet given injections of RS-2982 had reduced hepatic lipotoxicity, liver fibrosis, and body weight compared with mice given the vehicle. Agonists of RORA might be developed for treatment of NASH.

Cannabinoid-1 receptor regulates mitochondrial dynamics and function in renal proximal tubular cells.

AIMS: To evaluate the specific role of the endocannabinoid/cannabinoid type-1 (CB1 R) system in modulating mitochondrial dynamics in the metabolically active renal proximal tubular cells (RPTCs). MATERIALS AND METHODS: We utilized mitochondrially-targeted GFP in live cells (wild-type and null for the CB1 R) and electron microscopy in kidney sections of RPTC-CB1 R-/- mice and their littermate controls. In both in vitro and in vivo conditions, we assessed the ability of CB1 R agonism or fatty acid flux to modulate mitochondrial architecture and function. RESULTS: Direct stimulation of CB1 R resulted in mitochondrial fragmentation in RPTCs. This process was mediated, at least in part, by modulating the phosphorylation levels of the canonical fission protein dynamin-related protein 1 on both S637 and S616 residues. CB1 R-induced mitochondrial fission was associated with mitochondrial dysfunction, as documented by reduced oxygen consumption and ATP production, increased reactive oxygen species and cellular lactate levels, as well as a decline in mitochondrial biogenesis. Likewise, we documented that exposure of RPTCs to a fatty acid flux induced CB1 R-dependent mitochondrial fission, lipotoxicity and cellular dysfunction. CONCLUSIONS: CB1 R plays a key role in inducing mitochondrial fragmentation in RPTCs, leading to a decline in the organelle's function and contributing to the renal tubular injury associated with lipotoxicity and other metabolic diseases.

Modulation of Renal GLUT2 by the Cannabinoid-1 Receptor: Implications for the Treatment of Diabetic Nephropathy.

Altered glucose reabsorption via the facilitative glucose transporter 2 (GLUT2) during diabetes may lead to renal proximal tubule cell (RPTC) injury, inflammation, and interstitial fibrosis. These pathologies are also triggered by activating the cannabinoid-1 receptor (CB1R), which contributes to the development of diabetic nephropathy (DN). However, the link between CB1R and GLUT2 remains to be determined. Here, we show that chronic peripheral CB1R blockade or genetically inactivating CB1Rs in the RPTCs ameliorated diabetes-induced renal structural and functional changes, kidney inflammation, and tubulointerstitial fibrosis in mice. Inhibition of CB1R also downregulated GLUT2 expression, affected the dynamic translocation of GLUT2 to the brush border membrane of RPTCs, and reduced glucose reabsorption. Thus, targeting peripheral CB1R or inhibiting GLUT2 dynamics in RPTCs has the potential to treat and ameliorate DN. These findings may support the rationale for the clinical testing of peripherally restricted CB1R antagonists or the development of novel renal-specific GLUT2 inhibitors against DN.

Inhibition of somatostatin enhances the long-term metabolic outcomes of sleeve gastrectomy in mice.

OBJECTIVE: Bariatric surgery is an effective treatment to obesity, leading to weight loss and improvement in glycemia, that is characterized by hypersecretion of gastrointestinal hormones. However, weight regain and relapse of hyperglycemia are not uncommon. We set to identify mechanisms that can enhance gastrointestinal hormonal secretion following surgery to sustain weight loss. METHODS: We investigated the effect of somatostatin (Sst) inhibition on the outcomes of bariatric surgery using a mouse model of sleeve gastrectomy (SG). RESULTS: Sst knockout (sst-ko) mice fed with a calorie-rich diet gained weight normally and had a mild favorable metabolic phenotype compared to heterozygous sibling controls, including elevated plasma levels of GLP-1. Mathematical modeling of the feedback inhibition between Sst and GLP-1 showed that Sst exerts its maximal effect on GLP-1 under conditions of high hormonal stimulation, such as following SG. Obese sst-ko mice that underwent SG had higher levels of GLP-1 compared with heterozygous SG-operated controls. The SG-sst-ko mice regained less weight than controls and maintained lower glycemia months after surgery. Obese wild-type mice that underwent SG and were treated daily with a Sst receptor inhibitor for two months had higher GLP-1 levels, regained less weight, and improved metabolic profile compared to saline-treated SG-operated controls, and compared to inhibitor or saline-treated sham-operated obese mice. CONCLUSIONS: Our results suggest that inhibition of Sst signaling enhances the long-term favorable metabolic outcomes of bariatric surgery.

Hepatic targeting of the centrally active cannabinoid 1 receptor (CB1R) blocker rimonabant via PLGA nanoparticles for treating fatty liver disease and diabetes.

Over-activation of the endocannabinoid/CB1R system is a hallmark feature of obesity and its related comorbidities, most notably type 2 diabetes (T2D), and non-alcoholic fatty liver disease (NAFLD). Although the use of drugs that widely block the CB1R was found to be highly effective in treating all metabolic abnormalities associated with obesity, they are no longer considered a valid therapeutic option due to their adverse neuropsychiatric side effects. Here, we describe a novel nanotechnology-based drug delivery system for repurposing the abandoned first-in-class global CB1R antagonist, rimonabant, by encapsulating it in polymeric nanoparticles (NPs) for effective hepatic targeting of CB1Rs, enabling effective treatment of NAFLD and T2D. Rimonabant-encapsulated NPs (Rimo-NPs) were mainly distributed in the liver, spleen, and kidney, and only negligible marginal levels of rimonabant were found in the brain of mice treated by iv/ip administration. In contrast to freely administered rimonabant treatment, no CNS-mediated behavioral activities were detected in animals treated with Rimo-NPs. Chronic treatment of diet-induced obese mice with Rimo-NPs resulted in reduced hepatic steatosis and liver injury as well as enhanced insulin sensitivity, which were associated with enhanced cellular uptake of the formulation into hepatocytes. Collectively, we successfully developed a method of encapsulating the centrally acting CB1R blocker in NPs with desired physicochemical properties. This novel drug delivery system allows hepatic targeting of rimonabant to restore the metabolic advantages of blocking CB1R in peripheral tissues, especially in the liver, without the negative CB1R-mediated neuropsychiatric side effects.

Grape pomace reduces the severity of non-alcoholic hepatic steatosis and the development of steatohepatitis by improving insulin sensitivity and reducing ectopic fat deposition in mice.

While non-alcoholic fatty liver disease (NAFLD) represents the common cause of chronic liver disease, specific therapies are currently unavailable. The wine industry produces millions of tons of residue (pomace), which contains high levels of bioactive phytochemicals. The aim of this study was to clarify the potential benefits of grape pomace for the treatment of NAFLD at different levels of severity, and to clarify the mechanism of action. C57Bl/6 mice were given high fat diet (HFD) or western diet (WD) as models of obesity and hepatic steatosis or steatohepatitis, respectively, with or without pomace supplementation (50-250 mg/day). Pomace inhibited food intake, and reduced serum leptin and body weight gain. Ectopic fat deposition was reduced, while white adipose tissue mass was preserved. In addition, pomace improved glucose tolerance and insulin sensitivity, prevented the development of adipose tissue inflammation, and reduced hepatic steatosis. Higher expression of genes involved in fatty acids transport and oxidation was observed in adipose tissue, while lipogenic genes were attenuated in the liver of pomace-treated mice. In WD-fed mice, pomace reduced the severity of hepatic steatosis and inflammation and improved blood lipid profile, but was ineffective in reversing hepatic damage of advanced NASH. In conclusion, pomace improved insulin sensitivity and reduced ectopic fat deposition, leading to a healthier metabolic profile. Pomace may hold the potential as a supplement with beneficial health outcomes for the prevention and treatment of hepatic steatosis and other obesity-related pathologies.

Discriminant Analysis of Main Prognostic Factors Associated with Hemodynamically Significant PDA: Apgar Score, Silverman-Anderson Score, and NT-Pro-BNP Level.

Hemodynamically significant patent ductus arteriosus (hsPDA) in premature newborns is associated with a risk of PDA-related morbidities. Classification into risk groups may have a clinical utility in cases of suspected hsPDA to decrease the need for echocardiograms and unnecessary treatment. This prospective observational study included 99 premature newborns with extremely low body weight, who had an echocardiogram performed within the first three days of life. Discriminant analysis was utilized to find the best combination of prognostic factors for evaluation of hsPDA. We used binary logistic regression analysis to predict the relationship between parameters and hsPDA. The cohort's mean and standard deviation gestational age was 27.6 ± 2.55 weeks, the mean birth weight was 1015 ± 274 g. Forty-six (46.4%) infants had a PDA with a mean diameter of 2.78 mm. Median NT-pro-BNP levels were 17,600 pg/mL for infants with a PDA and 2773 pg/mL in the non-hsPDA group. The combination of prognostic factors of hsPDA in newborns of extremely low body weight on the third day of life was determined: NT-pro-BNP, Apgar score, Silverman-Anderson score (Se = 82%, Sp = 88%). A cut-off value of NT-pro-BNP of more than 8500 pg/mL can predict hsPDA (Se = 84%, Sp = 86%).

Sleeve Gastrectomy Suppresses Hepatic Glucose Production and Increases Hepatic Insulin Clearance Independent of Weight Loss.

Bariatric operations induce weight loss, which is associated with an improvement in hepatic steatosis and a reduction in hepatic glucose production. It is not clear whether these outcomes are entirely due to weight loss, or whether the new anatomy imposed by the surgery contributes to the improvement in the metabolic function of the liver. We performed vertical sleeve gastrectomy (VSG) on obese mice provided with a high-fat high-sucrose diet and compared them to diet and weight-matched sham-operated mice (WMS). At 40 days after surgery, VSG-operated mice displayed less hepatic steatosis compared with WMS. By measuring the fasting glucose and insulin levels in the blood vessels feeding and draining the liver, we showed directly that hepatic glucose production was suppressed after VSG. Insulin levels were elevated in the portal vein, and hepatic insulin clearance was elevated in VSG-operated mice. The hepatic expression of genes associated with insulin clearance was upregulated. We repeated the experiment in lean mice and observed that portal insulin and glucagon are elevated, but only insulin clearance is increased in VSG-operated mice. In conclusion, direct measurement of glucose and insulin in the blood entering and leaving the liver shows that VSG affects glucose and insulin metabolism through mechanisms independent of weight loss and diet.

Alleviation of a polyglucosan storage disorder by enhancement of autophagic glycogen catabolism.

This work employs adult polyglucosan body disease (APBD) models to explore the efficacy and mechanism of action of the polyglucosan-reducing compound 144DG11. APBD is a glycogen storage disorder (GSD) caused by glycogen branching enzyme (GBE) deficiency causing accumulation of poorly branched glycogen inclusions called polyglucosans. 144DG11 improved survival and motor parameters in a GBE knockin (Gbeys/ys ) APBD mouse model. 144DG11 reduced polyglucosan and glycogen in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic polyglucosan. At the cellular level, 144DG11 increased glycolytic, mitochondrial, and total ATP production. The molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. 144DG11 also enhanced mitochondrial activity and modulated lysosomal features as revealed by bioenergetic, image-based phenotyping and proteomics analyses. As an effective lysosomal targeting therapy in a GSD model, 144DG11 could be developed into a safe and efficacious glycogen and lysosomal storage disease therapy.

A Novel Indoline Derivative Ameliorates Diabesity-Induced Chronic Kidney Disease by Reducing Metabolic Abnormalities.

Both diabetes and obesity (diabesity) contribute significantly to the development of chronic kidney disease (CKD). In search of new remedies to reverse or arrest the progression of CKD, we examined the therapeutic potential of a novel compound, AN1284, in a mouse model of CKD induced by type 2 diabetes with obesity. Six-week-old BKS Cg-Dock 7m+/+ Leprdb/J mice with type 2 diabetes and obesity were treated with AN1284 (2.5 or 5 mg kg-1 per day) via micro-osmotic pumps implanted subcutaneously for 3 months. Measures included renal, pancreatic, and liver assessment as well as energy utilization. AN1284 improved kidney function in BSK-db/db animals by reducing albumin and creatinine and preventing renal inflammation and morphological changes. The treatment was associated with weight loss, decreased body fat mass, increased utilization of body fat toward energy, preservation of insulin sensitivity and pancreatic ß cell mass, and reduction of dyslipidemia, hepatic steatosis, and liver injury. This indoline derivative protected the kidney from the deleterious effects of hyperglycemia by ameliorating the metabolic abnormalities of diabetes. It could have therapeutic potential for preventing CKD in human subjects with diabesity.