Liver Biopsy Hydroxyproline Content Is a Diagnostic for Hepatocellular Carcinoma in Murine Models of Nonalcoholic Steatohepatitis.

There is increasing evidence that nonalcoholic steatohepatitis (NASH) is a risk factor for hepatocellular carcinoma (HCC) in the absence of cirrhosis, a phenomenon termed noncirrhotic HCC. Early diagnosis of HCC is critical to a favorable prognosis. We tested the hypothesis that hydroxyproline content of liver biopsy samples is diagnostic for HCC in murine models of NASH induced by diet or by diet and chemicals. The training set comprised mice fed a standard diet or a fast-food diet with or without administration of thioacetamide. At harvest, livers from the modified diet cohort exhibited NASH with a subset of NASH livers exhibiting HCC. Hydroxyproline content was measured in liver biopsy samples with tissue in the NASH+HCC cohort sampled from the remote, nontumor parenchyma. Plotting the receiver operating characteristics (ROC) with hydroxyproline as the continuous variable against the absence or presence of HCC yielded an area under ROC of 0.87, a threshold of >0.18 µg hydroxyproline/mg liver and sensitivity of 91% with a specificity of 83.3%. The use of liver hydroxyproline content as a diagnostic for HCC in a test set comprising healthy, NASH and NASH+HCC livers proved 87% accurate.

Stem cell-derived extracellular vesicles mitigate ageing-associated arterial stiffness and hypertension.

The prevalence of arterial stiffness and hypertension increases with age. This study investigates the effect of induced pluripotent mesenchymal stem cell-derived extracellular vesicles (EVs) on ageing-associated arterial stiffness and hypertension. EVs were collected and purified from induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSCs). Young and old male C57BL/6 mice were used. Mice in the EVs group were injected via tail vein once a week for four weeks (18 x 106 EVs/mouse/injection). Blood pressure (BP) was measured using the tail-cuff method and validated by direct cannulation. Pulse wave velocity (PWV) was measured using a Doppler workstation. PWV and BP were increased significantly in the old mice, indicating arterial stiffness and hypertension. Intravenous administration of EVs significantly attenuated ageing-related arterial stiffness and hypertension, while enhancing endothelium-dependent vascular relaxation and arterial compliance in the old EVs mice. Elastin degradation and collagen I deposition (fibrosis) were increased in aortas of the old mice, but EVs substantially improved ageing-associated structural remodelling. Mechanistically, EVs abolished downregulation of sirtuin type 1 (SIRT1), and endothelial nitric oxide synthase (eNOS) protein expression in aortas of the older mice. In cultured human aortic endothelial cells, EVs promoted the expression of SIRT1, AMP-activated protein kinase alpha (AMPKα), and eNOS. In conclusion, iPS-MSC-derived EVs attenuated ageing-associated vascular endothelial dysfunction, arterial stiffness, and hypertension, likely via activation of the SIRT1-AMPKα-eNOS pathway and inhibition of MMPs and elastase. Thus, EVs mitigate arterial ageing. This finding also sheds light into the therapeutic potential of EVs for ageing-related vascular diseases. ABBREVIATIONS: EV: Extracellular vesicles; iPS: induced pluripotent stem cell; MSC: mesenchymal stem cell; AMPKα: AMP activated protein kinase α; eNOS: endothelial nitric oxide synthase; Sirt1: sirtuin 1; JNC7: Seventh Report of the Joint National Committee; CVD: cardiovascular disease; PWV: pulse wave velocity; BP: blood pressure; SNP: sodium nitroprusside.

Glycerol-3-phosphate Acyltransferase1 Is a Model-Agnostic Node in Nonalcoholic Fatty Liver Disease: Implications for Drug Development and Precision Medicine.

Left untreated nonalcoholic fatty liver disease (NAFLD) can progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. The observed failure of clinical trials in NASH may suggest that current model systems do not fully recapitulate human disease, and/or hallmark pathological features of NASH may not be driven by the same pathway in every animal model let alone in each patient. Identification of a model-agnostic disease-associated node can spur the development of effective drugs for the treatment of liver disease. Glycerol-3-phosphate acyltransferase1 (GPAT1) plays a pivotal role in lipid accumulation by shunting fats away from oxidation. In the present study, hepatic GPAT1 expression was evaluated in three etiologically different models of NAFLD. Compared to the sham cohort, hepatic GPAT1 mRNA levels were elevated by ∼5-fold in steatosis and NASH with fibrosis with immunofluorescent staining revealing increased GPAT1 in the fatty liver. A significant and direct correlation (r = 0.88) was observed between hepatic GPAT1 mRNA expression and severity of the liver disease. Picrosirius red staining revealed a logarithmic relation between hepatic GPAT1 mRNA expression and scar. These data suggest that hepatic GPAT1 is an early disease-associated model-agnostic node in NAFLD and form the basis for the development of a potentially successful therapeutic against NASH.

Prevention of lipopolysaccharide-induced CD11b+ immune cell infiltration in the kidney: role of AT2 receptors.

Immune cell infiltration plays a central role in mediating endotoxemic acute kidney injury (AKI). Recently, we have reported the anti-inflammatory and reno-protective role of angiotensin-II type-2 receptor (AT2R) activation under chronic low-grade inflammatory condition in the obese Zucker rat model. However, the role of AT2R activation in preventing lipopolysaccharide (LPS)-induced early infiltration of immune cells, inflammation and AKI is not known. Mice were treated with AT2R agonist C21 (0.3 mg/kg), with and without AT2R antagonist PD123319 (5 mg/kg) prior to or concurrently with LPS (5 mg/kg) challenge. Prior-treatment with C21, but not concurrent treatment, significantly prevented the LPS-induced renal infiltration of CD11b+ immune cells, increase in the levels of circulating and/or renal chemotactic cytokines, particularly interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) and markers of renal dysfunction (blood urea nitrogen and albuminuria), while preserving anti-inflammatory interleukin-10 (IL-10) production. Moreover, C21 treatment in the absence of LPS increased renal and circulating IL-10 levels. To investigate the role of IL-10 in a cross-talk between epithelial cells and monocytes, we performed in vitro conditioned media (CM) studies in human kidney proximal tubular epithelial (HK-2) cells and macrophages (differentiated human monocytes, THP-1 cells). These studies revealed that the conditioned-media derived from the C21-treated HK-2 cells reduced LPS-induced THP-1 tumor necrosis factor-α (TNF-α) production via IL-10 originating from HK-2 cells. Our findings suggest that prior activation of AT2R is prophylactic in preventing LPS-induced renal immune cell infiltration and dysfunction, possibly via IL-10 pathway.

Deep Learning in Drug Discovery and Medicine; Scratching the Surface.

The practice of medicine is ever evolving. Diagnosing disease, which is often the first step in a cure, has seen a sea change from the discerning hands of the neighborhood physician to the use of sophisticated machines to use of information gleaned from biomarkers obtained by the most minimally invasive of means. The last 100 or so years have borne witness to the enormous success story of allopathy, a practice that found favor over earlier practices of medical purgatory and homeopathy. Nevertheless, failures of this approach coupled with the omics and bioinformatics revolution spurred precision medicine, a platform wherein the molecular profile of an individual patient drives the selection of therapy. Indeed, precision medicine-based therapies that first found their place in oncology are rapidly finding uses in autoimmune, renal and other diseases. More recently a new renaissance that is shaping everyday life is making its way into healthcare. Drug discovery and medicine that started with Ayurveda in India are now benefiting from an altogether different artificial intelligence (AI)-one which is automating the invention of new chemical entities and the mining of large databases in health-privacy-protected vaults. Indeed, disciplines as diverse as language, neurophysiology, chemistry, toxicology, biostatistics, medicine and computing have come together to harness algorithms based on transfer learning and recurrent neural networks to design novel drug candidates, a priori inform on their safety, metabolism and clearance, and engineer their delivery but only on demand, all the while cataloging and comparing omics signatures across traditionally classified diseases to enable basket treatment strategies. This review highlights inroads made and being made in directed-drug design and molecular therapy.

Supervised learning reveals circulating biomarker levels diagnostic of hepatocellular carcinoma in a clinically relevant model of non-alcoholic steatohepatitis; An OAD to NASH.

Although cirrhosis is a key risk factor for the development of hepatocellular carcinoma (HCC), mounting evidence indicates that in a subset of patients presenting with non-alcoholic steatohepatitis (NASH) HCC manifests in the absence of cirrhosis. Given the sheer size of the ongoing non-alcoholic fatty liver disease (NAFLD) epidemic and the dismal prognosis associated with late-stage primary liver cancer there is an urgent need for HCC surveillance in the NASH population. Using serum levels of HCC biomarkers as vectors and biopsy-proven HCC or no HCC as outputs / binary classifier, a supervised learning campaign was undertaken to develop a minimally invasive technique for making a diagnosis of HCC in a clinically relevant model of NASH. Adult mice randomized to control diet or a fast food diet (FFD) were followed for up to 14 mo and serum level of a panel of HCC-relevant biomarkers was compared with liver biopsies at 3 and 14 mo. Both NAFLD Activity Score (NAS) and hepatic hydroxyproline content were elevated at 3 and 14 mo on FFD. Picrosirius red staining of liver sections revealed a filigree pattern of fibrillar collagen deposition with no cirrhosis at 14 mo on FFD. Nevertheless, 46% of animals bore one or more tumors on their livers confirmed as HCC in hematoxylin-eosin-stained liver sections. In this training set, receiver operating characteristic (ROC) curves analysis for serum levels of the HCC biomarkers osteopontin (OPN), alpha-fetoprotein (AFP) and Dickkopf-1 (DKK1) returned concordance-statistic/area under ROC curve of ≥ 0.89. Serum levels of OPN (threshold, 218 ng/mL; sensitivity, 82%; specificity, 86%), AFP (136 ng/mL; 91%; 97%) and DKK1 (2.4 ng/mL; 82%; 81%) diagnostic for HCC were confirmed in a test set comprising mice on control diet or FFD and mice subjected to hepatic ischemia-reperfusion injury. These data suggest that levels of circulating OPN, AFP and DKK1 can be used to make a diagnosis of HCC in a clinically relevant model of NASH.

Angiotensin II Type 2 Receptor and Receptor Mas Are Colocalized and Functionally Interdependent in Obese Zucker Rat Kidney.

The actions of angiotensin II type 2 receptor (AT2R) and the receptor Mas (MasR) are complex but show similar pronatriuretic function; particularly, AT2R expression and natriuretic function are enhanced in obese/diabetic rat kidney. In light of some reports suggesting a potential positive interaction between these receptors, we tested hypothesis that renal AT2R and MasR physically interact and are interdependent to stimulate cell signaling and promote natriuresis in obese rats. We found that infusion of AT2R agonist C21 in obese Zucker rats (OZR) increased urine flow and urinary Na excretion which were attenuated by simultaneous infusion of the AT2R antagonist PD123319 or the MasR antagonist A-779. Similarly, infusion of MasR agonist Ang-(1-7) in OZR increased urine flow and urinary Na excretion, which were attenuated by simultaneous infusion of A-779 or PD123319. Experiment in isolated renal proximal tubules of OZR revealed that both the agonists C21 and Ang-(1-7) stimulated NO which was blocked by either of the receptor antagonists. Dual labeling of AT2R and MasR in OZR kidney sections and human proximal tubule epithelial cells showed that AT2R and MasR are colocalized. The AT2R also coimmunoprecipitated with MasR in cortical homogenate of OZR. Immunoblotting of cortical homogenate cross-linked with zero-length oxidative (sulfhydryl groups) cross-linker cupric-phenanthroline revealed a shift of AT2R and MasR bands upward with overlapping migration for their complexes which were sensitive to the reducing ß-mercaptoethanol, suggesting involvement of -SH groups in cross-linking. Collectively, the study reveals that AT2R and MasR are colocalized and functionally interdependent in terms of stimulating NO and promoting diuretic/natriuretic response.

Monocrotaline-Induced Pulmonary Hypertension Involves Downregulation of Antiaging Protein Klotho and eNOS Activity.

The objective of this study is to investigate whether stem cell delivery of secreted Klotho (SKL), an aging-suppressor protein, attenuates monocrotaline-induced pulmonary vascular dysfunction and remodeling. Overexpression of SKL in mesenchymal stem cells (MSCs) was achieved by transfecting MSCs with lentiviral vectors expressing SKL-green fluorescent protein (GFP). Four groups of rats were treated with monocrotaline, whereas an additional group was given saline (control). Three days later, 4 monocrotaline-treated groups received intravenous delivery of nontransfected MSCs, MSC-GFP, MSC-SKL-GFP, and PBS, respectively. Ex vivo vascular relaxing responses to acetylcholine were diminished in small pulmonary arteries (PAs) in monocrotaline-treated rats, indicating pulmonary vascular endothelial dysfunction. Interestingly, delivery of MSCs overexpressing SKL (MSC-SKL-GFP) abolished monocrotaline-induced pulmonary vascular endothelial dysfunction and PA remodeling. Monocrotaline significantly increased right ventricular systolic blood pressure, which was attenuated significantly by MSC-SKL-GFP, indicating improved PA hypertension. MSC-SKL-GFP also attenuated right ventricular hypertrophy. Nontransfected MSCs slightly, but not significantly, improved PA hypertension and pulmonary vascular endothelial dysfunction. MSC-SKL-GFP attenuated monocrotaline-induced inflammation, as evidenced by decreased macrophage infiltration around PAs. MSC-SKL-GFP increased SKL levels, which rescued the downregulation of SIRT1 (Sirtuin 1) expression and endothelial NO synthase (eNOS) phosphorylation in the lungs of monocrotaline-treated rats. In cultured endothelial cells, SKL abolished monocrotaline-induced downregulation of eNOS activity and NO levels and enhanced cell viability. Therefore, stem cell delivery of SKL is an effective therapeutic strategy for pulmonary vascular endothelial dysfunction and PA remodeling. SKL attenuates monocrotaline-induced PA remodeling and PA smooth muscle cell proliferation, likely by reducing inflammation and restoring SIRT1 levels and eNOS activity.

Activation of SIRT1 Attenuates Klotho Deficiency-Induced Arterial Stiffness and Hypertension by Enhancing AMP-Activated Protein Kinase Activity.

Arterial stiffness is an independent risk factor for stroke and myocardial infarction. This study was designed to investigate the role of SIRT1, an important deacetylase, and its relationship with Klotho, a kidney-derived aging-suppressor protein, in the pathogenesis of arterial stiffness and hypertension. We found that the serum level of Klotho was decreased by ≈45% in patients with arterial stiffness and hypertension. Interestingly, Klotho haplodeficiency caused arterial stiffening and hypertension, as evidenced by significant increases in pulse wave velocity and blood pressure in Klotho-haplodeficient (KL+/-) mice. Notably, the expression and activity of SIRT1 were decreased significantly in aortic endothelial and smooth muscle cells in KL+/- mice, suggesting that Klotho deficiency downregulates SIRT1. Treatment with SRT1720 (15 mg/kg/d, IP), a specific SIRT1 activator, abolished Klotho deficiency-induced arterial stiffness and hypertension in KL+/- mice. Klotho deficiency was associated with significant decreases in activities of AMP-activated protein kinase α (AMPKα) and endothelial NO synthase (eNOS) in aortas, which were abolished by SRT1720. Furthermore, Klotho deficiency upregulated NADPH oxidase activity and superoxide production, increased collagen expression, and enhanced elastin fragmentation in the media of aortas. These Klotho deficiency-associated changes were blocked by SRT1720. In conclusion, this study provides the first evidence that Klotho deficiency downregulates SIRT1 activity in arterial endothelial and smooth muscle cells. Pharmacological activation of SIRT1 may be an effective therapeutic strategy for arterial stiffness and hypertension.

Grape Powder Improves Age-Related Decline in Mitochondrial and Kidney Functions in Fischer 344 Rats.

We examined the effects and mechanism of grape powder- (GP-) mediated improvement, if any, on aging kidney function. Adult (3-month) and aged (21-month) Fischer 344 rats were treated without (controls) and with GP (1.5% in drinking water) and kidney parameters were measured. Control aged rats showed higher levels of proteinuria and urinary kidney injury molecule-1 (KIM-1), which decreased with GP treatment in these rats. Renal protein carbonyls (protein oxidation) and gp (91phox) -NADPH oxidase levels were high in control aged rats, suggesting oxidative stress burden in these rats. GP treatment in aged rats restored these parameters to the levels of adult rats. Moreover, glomerular filtration rate and sodium excretion were low in control aged rats suggesting compromised kidney function, which improved with GP treatment in aged rats. Interestingly, low renal mitochondrial respiration and ATP levels in control aged rats were associated with reduced levels of mitochondrial biogenesis marker MtTFA. Also, Nrf2 proteins levels were reduced in control aged rats. GP treatment increased levels of MtTFA and Nrf2 in aged rats. These results suggest that GP by potentially regulating Nrf2 improves aging mitochondrial and kidney functions.

Angiotensin type 2 receptor null mice express reduced levels of renal angiotensin II type 2 receptor/angiotensin (1-7)/Mas receptor and exhibit greater high-fat diet-induced kidney injury.

INTRODUCTION: Renin-angiotensin system (RAS) components exert diverse physiological functions and have been sub-grouped into deleterious angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/angiotensin type 1 receptor (AT1R) and protective ACE2/angiotensin (1-7) (Ang-(1-7))/Mas receptor (MasR) axes. We have reported that chronic activation of angiotensin type 2 receptor (AT2R) alters RAS components and provides protection against obesity-related kidney injury. MATERIALS AND METHODS: We utilized AT2R knockout (AT2KO) mice in this study and evaluated the renal expression of various RAS components and examined the renal injury after placing these mice on high fat diet (HFD) for 16 weeks. RESULTS: The cortical ACE2 activity and MasR expression were significantly decreased in AT2KO mice compared to wild type (WT) mice. LC/MS analysis revealed an increase in renal Ang II levels and a decrease in Ang-(1-7) levels in AT2KO mice. Cortical expression of ACE and AT1R was increased but renin activity remained unchanged in AT2KO compared with WT mice. WT mice fed HFD exhibited increased systolic blood pressure, higher indices of kidney injury, mesangial matrix expansion score, and microalbuminuria, which were further increased in AT2KO mice. CONCLUSION: This study suggests that deletion of AT2R decreases the expression of the beneficial ACE2/Ang-(1-7)/MasR and increases the deleterious ACE/Ang II/AT1R axis of the renal RAS in mice. Further, AT2KO mice are more susceptible to HFD-induced renal injury.

Angiotensin II Type 2-Receptor Agonist C21 Reduces Proteinuria and Oxidative Stress in Kidney of High-Salt-Fed Obese Zucker Rats.

Oxidative and nitrosative stress have been implicated in high-sodium diet (HSD)-related hypertensive renal injury. In this study, we investigated angiotensin II type 2-receptor-mediated renoprotection in obese Zucker rats fed HSD. Obese Zucker rats were fed normal sodium diet or HSD 4%, for 14 days, with/without angiotensin II type 2-receptor agonist C21, delivered subcutaneously via osmotic pump, 1 mg/kg per day. Compared with normal sodium diet controls, HSD rats exhibited increase in cortical nicotinamide adenine dinucleotide phosphate oxidase activity, urinary H2O2, and 8-isoprostanes, which were associated with severe glomerulosclerosis, interstitial fibrosis, decline in estimated glomerular filtration rate, and an increase in urinary leak and activity of N-acetyl-ß-D-glucosaminidase, a lysosomal enzyme and a marker of tubular damage. These changes were improved by C21 treatment. Cortical expression of endothelial nitric oxide synthase, phospho-endothelial nitric oxide synthase (Ser(1177)), and plasma nitrites were reduced after HSD intake, whereas nitrosative stress (3-nitrotyrosine) and enzymatic defense (superoxide dismutase-to-catalase activity) remained unaltered. However, C21 preserved plasma nitrites in HSD-fed obese Zucker rat. C21 treatment reduced protein-to-creatinine, albumin-to-creatinine, as well as fractional excretion of protein and albumin in HSD-fed obese Zucker rat, which is independent of changes in protein recycling receptors, megalin, and cubilin. HSD intake also altered renal excretory and reabsorptive capacity as evident by elevated plasma urea nitrogen-to-creatinine and fractional excretion of urea nitrogen, and reduced urine-to-plasma creatinine, which were modestly, but insignificantly, improved by C21 treatment. Together results demonstrate that angiotensin II type 2-receptor activation protects against HSD-induced kidney damage in obesity plausibly by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and rescuing nitrites.

Grape powder treatment prevents anxiety-like behavior in a rat model of aging.

Earlier, we have reported that grape powder (GP) treatment prevented pharmacologic and psychological stress-induced anxiety-like behavior and memory impairment in rats. Protective effects of GP were attributed to its antioxidant effects. In this study, we tested the hypothesis that age-associated behavioral and cognitive deficits such as anxiety and memory impairment will be ameliorated with GP treatment. Using a National Institute of Aging recommended rodent model of aging, we examined a potentially protective role of antioxidant-rich GP in age-associated anxiety-like behavior and memory impairment. Male Fischer 344 rats were randomly assigned into 4 groups: young rats (3 months old) provided with tap water or with 15 g/L GP dissolved in tap water for 3 weeks, aged rats (21 months old) provided with tap water or with GP-treated tap water for 3 weeks (AG-GP). Anxiety-like behavior was significantly greater in aged rats compared with young rats, GP-treated young rats, or aged control rats (P < .05). Also, GP treatment prevented age-induced anxiety-like behavior in AG-GP rats (P < .05). Neither short-term nor long-term age-associated memory deficits improved with GP treatment in AG-GP rats. Furthermore, aged rats showed increased level of physiological stress (corticosterone) and increased oxidative stress in the plasma (8-isoprostane) as well as in selected brain areas (protein carbonylation). Grape powder treatment prevented age-induced increase in corticosterone levels and plasma 8-isoprostane levels in aged rats (P < .05), whereas protein carbonylation was recovered in the amygdala region only (P < .05). Grape powder by regulating oxidative stress ameliorates age-induced anxiety-like behavior in rats, whereas age-associated memory deficits seem unaffected with GP treatment.

Angiotensin AT2 receptor agonist prevents salt-sensitive hypertension in obese Zucker rats.

High-sodium intake is a risk factor for the pathogenesis of hypertension, especially in obesity. The present study is designed to investigate whether angiotensin type 2 receptor (AT2R) activation with selective agonist C21 prevents high-sodium diet (HSD)-induced hypertension in obese animals. Male obese rats were treated with AT2R agonist C21 (1 mg·kg(-1)·day(-1), oral) while maintained on either normal-sodium diet (NSD; 0.4%) or HSD (4%) for 2 wk. Radiotelemetric recording showed a time-dependent increase in systolic blood pressure in HSD-fed obese rats, being maximal increase (∼27 mmHg) at day 12 of the HSD regimen. C21 treatment completely prevented the increase in blood pressure of HSD-fed rats. Compared with NSD controls, HSD-fed obese rats had greater natriuresis/diuresis and urinary levels of nitrates, and these parameters were further increased by C21 treatment. Also, C21 treatment improved glomerular filtration rate in HSD-fed rats. HSD-fed rats expressed higher level of cortical ANG II, which was reduced to 50% by C21 treatment. HSD feeding and/or C21 treatment had no effects on cortical renin activity and the expression of angiotensin-converting enzyme (ACE) and chymase, which are ANG II-producing enzymes. However, ANG(1-7) concentration and ACE2 activity in the renal cortex were reduced by HSD feeding, and C21 treatment rescued both the parameters. Also, C21 treatment reduced the cortical expression of AT1R in HSD-fed rats, but had no effect of AT2R expression. We conclude that chronic treatment with the AT2R agonist C21 prevents salt-sensitive hypertension in obese rats, and a reduction in the renal ANG II/AT1R and enhanced ACE2/ANG(1-7) levels may play a potential role in this phenomenon.

Chronic angiotensin AT2R activation prevents high-fat diet-induced adiposity and obesity in female mice independent of estrogen.

OBJECTIVE: Obesity is a known risk factor for various metabolic disorders and cardiovascular diseases. Recently we demonstrated that female angiotensin AT2 receptor (AT2R) knockout mice on high-fat diet (HFD) had higher body weight and adiposity with a parallel reduction in estrogen (17,ß-estradiol/E2). The present study investigated whether the anti-adiposity effects of the AT2R are estrogen-dependent in female mice. METHODS: Female C57BL/6 ovary-intact (Ovi) mice were treated with an AT2R agonist (C21, 0.3 mg/kg, daily i.p.). Ovariectomized (Ovx) mice, supplemented with E2 (5 µg/day, pellets implanted subcutaneously), were treated with an AT2R agonist (C21, 0.3 mg/kg, daily i.p.) or vehicle. After 4-days of pre-treatment with C21, Ovi and Ovx mice were placed on either normal diet (ND) or HFD while the C21 treatment continued for the next 10 days. For a long-term study, Ovi mice were placed on HFD and treated with C21 for 12 weeks. RESULTS: Ovi mice fed the HFD had increased parametrial white adipose tissue (pWAT) weight, plasma free fatty acid and triglycerides compared to Ovi mice on ND. Ovariectomy alone caused similar changes in these parameters which were further increased by HFD-feeding. C21 treatment attenuated these HFD-induced changes in Ovi as well as Ovx mice. HFD also, increased the liver/body-weight ratio and decreased the liver expression of the ß-oxidation enzyme, carnitine palmitoyltransferase 1 (CPT1-A). C21 treatment attenuated these changes as well. The long-term C21 treatment of Ovi mice lowered the HFD-induced body weight gain, increase in pWAT weight, parametrial adipocyte size and hyperinsulinemia induced by HFD. Finally, HFD drastically reduced urinary estrogen and the beneficial metabolic changes in response to C21-treatment occurred without significantly increasing urinary estrogen. CONCLUSION: We suggest that the pharmacological activation of AT2R by the agonist C21 reduces adiposity and body weight gain independent of estrogen in female mice. Improvement in fatty acid metabolism is a potential mechanism by which the AT2R exerts anti-adiposity effects.

Estimation of angiotensin peptides in biological samples by LC/MS method.

The low abundance of angiotensin peptides in biological tissues such as the kidney cortex, adipose tissue, urine and plasma makes their detection and quantification a challenge. A few available methods used to quantify these peptides involve lengthy processes of sample preparation and are hardly quantitative. Here, we report a mass spectrometry approach for quantifying angiotensin peptides [Ang II, Ang-(1-7)] in the kidney cortex, epididymal white adipose tissue (eWAT), urine and plasma of male mice. Tissue homogenates, urine and plasma samples were solid-phase extracted with C18 Sep-Pak cartridges and eluted off proteinaceous compounds. These extracted peptide samples were separated on C18 column with a linear acetonitrile gradient and detected by Q-ToF mass analyzer in ESI+-MS ion mode based on their retention time, accurate mass measurement of peptides, the isotope pattern of doubly charged molecular ion, and quantitation of peak area (or ion count) when referencing to the angiotensin peptide standards. The lower limit of quantitation for each angiotensin peptide was 10 pgmg-1 with the percent recovery at 100.6%. The intra-batch precision for Ang-(1-7) and Ang II were 24.0 and 12.7%, accuracy 84.0-123.0% and 100.2-116.0% respectively. Using this method, we determined the levels of Ang II and Ang-(1-7) in the kidney cortex, eWAT, urine and plasma. Quantification of angiotensin peptides could help target subtle therapeutics changes against pathophysiological conditions such as obesity, kidney disease and hypertension.

Chronic AT2 receptor activation increases renal ACE2 activity, attenuates AT1 receptor function and blood pressure in obese Zucker rats.

Abnormal regulation of the renin angiotensin system such as enhanced renal AT1R function and reduced ACE2 activity contributes to obesity-related hypertension. Here, we tested whether long-term AT2R activation affects renal function in obesity using lean and obese Zucker rats treated with the AT2R agonist CGP42112A for 2 weeks. This caused blood pressure to decrease by 13 mm Hg, which was associated with increased urinary sodium excretion in the obese rats. Cortical ACE2 expression and activity, the Mas receptor (MasR), and its ligand angiotensin-(1-7) were all increased in CGP-treated obese compared with control rats. Candesartan-induced natriuresis, a measure of AT1R function, was reduced but cortical AT1R expression and angiotensin II levels were similar in CGP-treated obese compared with control rats. Renin and AT2R expression in obese rats was not affected by CGP treatment. In HK-2 cells in vitro, CGP treatment caused increased ACE2 activity and MasR levels but decreased AT1R levels and renin activity. Thus, long-term AT2R activation shifts the opposing arms of renin angiotensin system and contributes to natriuresis and blood pressure reduction in obese animals. Our study highlights the importance of AT2R as a target for treating obesity-related hypertension.

Proximal tubule angiotensin AT2 receptors mediate an anti-inflammatory response via interleukin-10: role in renoprotection in obese rats.

The angiotensin type 2 receptor (AT2R) has been shown to lower inflammation in the kidney. However, the role of the anti-inflammatory cytokine interleukin (IL)-10 in AT2R-mediated attenuation of inflammation has not been elucidated. We hypothesized that AT2R activation is renoprotective by directly increasing the levels of anti-inflammatory cytokine IL-10 in the kidney via nitric oxide (NO) signaling. For in vitro studies, the human proximal tubule epithelial cell-line (human kidney-2 [HK-2]) was activated with lipopolysaccharide (10 µg/mL) and AT2R agonist C21 (1 µmol/L) for 24 hours, and media cytokine levels were assessed. Lipopolysaccharide modestly downregulated AT2R expression. Treatment with C21 lowered lipopolysaccharide-induced levels of both tumor necrosis factor-α and IL-6, but increased IL-10 levels. Treatment with neutralizing IL-10 antibody (1 µg/mL) or NO synthase inhibitor L-NAME (1 mmol/L) abolished this effect. For in vivo studies, prehypertensive obese Zucker rats and age-matched lean Zucker rats were treated for 2 weeks with C21 (300 µg/kg per day, IP) and AT2R antagonist (PD123319; 50 µg/kg per minute, SC infusion). Compared with lean Zucker rats, obese Zucker rats had higher levels of renal AT2R expression, tumor necrosis factor-α, and IL-6. C21 treatment decreased levels of tumor necrosis factor-α by 75% and IL-6 by 60%. Conversely, PD treatment lowered the renal IL-10 levels in obese Zucker rats by ≈60%. Renal morphometry revealed increased mesangial matrix expansion and glomerular macrophage infiltration, which was improved by C21 treatment in obese Zucker rats. Our findings suggest that proximal tubule AT2R activation is anti-inflammatory by increasing IL-10 production, which is largely NO dependent and thus offers renoprotection by preventing early inflammation-induced renal injury in obesity.

High Na intake increases renal angiotensin II levels and reduces expression of the ACE2-AT(2)R-MasR axis in obese Zucker rats.

High sodium intake is known to regulate the renal renin-angiotensin system (RAS) and is a risk factor for the pathogenesis of obesity-related hypertension. The complex nature of the RAS reveals that its various components may have opposing effects on natriuresis and blood pressure regulation. We hypothesized that high sodium intake differentially regulates and shifts a balance between opposing components of the renal RAS, namely, angiotensin-converting enzyme (ACE)-ANG II-type 1 ANG II receptor (AT(1)R) vs. AT(2)-ACE2-angiotensinogen (Ang) (1-7)-Mas receptor (MasR), in obesity. In the present study, we evaluated protein and/or mRNA expression of angiotensinogen, renin, AT(1A/B)R, ACE, AT(2)R, ACE2, and MasR in the kidney cortex following 2 wk of a 8% high-sodium (HS) diet in lean and obese Zucker rats. The expression data showed that the relative expression pattern of ACE and AT(1B)R increased, renin decreased, and ACE2, AT(2)R, and MasR remained unaltered in HS-fed lean rats. On the other hand, HS intake in obese rats caused an increase in the cortical expression of ACE, a decrease in ACE2, AT(2)R, and MasR, and no changes in renin and AT(1)R. The cortical levels of ANG II increased by threefold in obese rats on HS compared with obese rats on normal salt (NS), which was not different than in lean rats. The HS intake elevated mean arterial pressure in obese rats (27 mmHg) more than in lean rats (16 mmHg). This study suggests that HS intake causes a pronounced increase in ANG II levels and a reduction in the expression of the ACE2-AT(2)R-MasR axis in the kidney cortex of obese rats. We conclude that such changes may lead to the potentially unopposed function of AT(1)R, with its various cellular and physiological roles, including the contribution to the pathogenesis of obesity-related hypertension.