Angiotensin-II type 2 receptor-mediated renoprotection is independent of receptor Mas in obese Zucker rats fed high-sodium diet.

The consumption of a high-sodium diet (HSD) is injurious and known to elevate blood pressure (BP), especially in obesity. Acute infusion studies depict a functional interdependency between angiotensin-II type 2 receptor (AT2R) and receptor Mas (MasR). Hence, we hypothesize that the subacute blockade of MasR should reverse AT2R-mediated renoprotection in obese Zucker rats (OZRs). Male OZRs were fed an HSD (for 14 days) and treated with the AT2R agonist C21 (100 ng/min) without or with a MasR antagonist A779 (1,000 ng/min). The indices of oxidative stress, proteinuria, kidney injury, and BP were measured before and after, along with the terminal measurements of an array of inflammatory and kidney injury markers. The HSD significantly decreased the estimated glomerular filtration rate and urinary osmolality and increased thirst, diuresis, natriuresis, kaliuresis, plasma creatinine, urinary excretion of H2O2, proteinuria, renal expression and urinary excretion of kidney injury markers (NGAL and KIM-1), and BP indexes. The HSD feeding showed early changes in the renal expression of CRP, ICAM-1, and galectin-1. The C21 treatment prevented these pathological changes. The MasR antagonist A779 attenuated C21-mediated effects on the urinary excretion and renal expression of NGAL and oxidative stress in the absence of inflammation and BP changes. Overall, we conclude that the subacute functional interactions between AT2R and MasR are weak or transient and that the beneficial effects of AT2R activation are independent of the MasR blockade in the kidney of male obese rats fed an HSD.

Angiotensin II Type 2 Receptor: A Target for Protection Against Hypertension, Metabolic Dysfunction, and Organ Remodeling.

The renin-angiotensin system is of vital significance not only in the maintenance of blood pressure but also because of its role in the pathophysiology of different organ systems in the body. Of the 2 Ang II (angiotensin II) receptors, the AT1R (Ang II type 1 receptor) has been extensively studied for its role in mediating the classical functions of Ang II, including vasoconstriction, stimulation of renal tubular sodium reabsorption, hormonal secretion, cell proliferation, inflammation, and oxidative stress. The other receptor, AT2R (Ang II type 2 receptor), is abundantly expressed in both immune and nonimmune cells in fetal tissue. However, its expression is increased under pathological conditions in adult tissues. The role of AT2R in counteracting AT1R function has been discussed in the past 2 decades. However, with the discovery of the nonpeptide agonist C21, the significance of AT2R in various pathologies such as obesity, hypertension, and kidney diseases have been examined. This review focuses on the most recent findings on the beneficial effects of AT2R by summarizing both gene knockout studies as well as pharmacological studies, specifically highlighting its importance in blood pressure regulation, obesity/metabolism, organ protection, and relevance in the treatment of coronavirus disease 2019 (COVID-19).

Combining Neprilysin Inhibitor With AT2R Agonist Is Superior to Combination With AT1R Blocker in Providing Reno-Protection in Obese Rats.

Clinical use of the combination therapy of the neprilysin inhibitor sacubitril and angiotensin II type 1 receptor blocker valsartan is known to be associated with albuminuria. Albuminuria is both a risk factor for and an indicator of kidney injury. Earlier work from our laboratory reported that the agonist of angiotensin II type 2 receptor Compound 21 (C21) prevents proteinuria, albuminuria, and is reno-protective in obese Zucker rats fed high salt diet (HSD). Thus, we hypothesized that sacubitril/C21 combination provides superior reno-protection compared to sacubitril/valsartan. Male obese Zucker rats 10-11 weeks old were treated daily with vehicle, sacubitril + C21, or sacubitril + valsartan while fed HSD for 16 days. HSD-feeding caused kidney dysfunction, evident by significant increases in urinary protein, osteopontin, and cystatin C. HSD-feeding lowered plasma cystatin C and creatinine concentrations suggestive of hyperfiltration, which was not affected by either treatment. Unlike sacubitril/valsartan, sacubitril/C21 treatment significantly decreases proteinuria, albuminuria, the expression of nephrin, and kidney weight, independent of hyperfiltration, compared with HSD alone. Moreover, sacubitril/valsartan therapy increased plasma renin and did not prevent HSD-induced increases in renal angiotensin II, while sacubitril/C21 completely prevented these changes. Together, this study suggests that sacubitril/C21 afforded superior reno-protection compared to sacubitril/valsartan therapy in high salt-fed obese Zucker rats.

Emerging Role of Angiotensin AT2 Receptor in Anti-Inflammation: An Update.

The hyperactive RAS and inflammation are closely associated. The angiotensin-II/AT1R axis of the RAS has been explored extensively for its role in inflammation and a plethora of pathological conditions. Understanding the role of AT2R in inflammation is an emerging area of research. The AT2R is expressed on a variety of immune and non-immune cells, which upon activation triggers the release of a host of cytokines and has multiple effects that coalesce to anti-inflammation and prevents maladaptive repair. The anti-inflammatory outcomes of AT2R activation are linked to its well-established signaling pathways involving formation of nitric oxide and activation of phosphatases. Collectively, these effects promote cell survival and tissue function. The consideration of AT2R as a therapeutic target requires further investigations.

Modification by Ethanol and Taurine, Singly and in Combination, of Changes in Indices of Renal Dysfunction Caused by Diabetes in Rats.

The present study was carried out in diabetic rats to examine the effects of ethanol (EtOH) and taurine (TAU), singly and in combination, in reducing the changes of laboratory test values indicating renal dysfunction. For this purpose, male Sprague-Dawley rats, 250-280 g in weight and in groups of 6, were made diabetic with a single, 60 mg/kg intraperitoneal dose of streptozotocin in 10 mM citrate buffer pH 4.5. On day 15 and for the remaining 14 days of the study, the diabetic rats (a) started to drink 5% EtOH in place of water, (b) received a single daily 2.4 mM/kg oral dose of TAU or (c) were allowed to drink 5% EtOH after receiving a dose of TAU. Starting from day 28 and ending on day 29, a 24 h urine sample was collected, its volume was measured, and then used to measure glucose (GLC), total protein (TP) and electrolytes (Na+, K+, Ca++, Mg++). Blood samples collected immediately thereafter via cardiac puncture were processed for the plasma fractions which were analyzed for their creatinine (CRT) and urea nitrogen (UN) contents. In comparison to normal (control) rats, diabetic ones showed a higher output of urine (+5.6-fold), a massive increase in plasma GLC (+473%), passed more GLC (+73.8-fold) and TP (+8.2-fold) in the urine, showed higher plasma CRT (+241%) and UN (+74%) levels, a lower plasma UN/CRT ratio (-47%) and a greater output of electrolytes in the urine (by at least twofold). By themselves both EtOH and TAU were found to markedly lower the effects of diabetes, with EtOH generally appearing more effective than TAU. However, the concurrent availability of EtOH and TAU was found to be more protective than either treatment alone.

The Effect of Taurine and Its Immediate Homologs on Diabetes-Induced Oxidative Stress in the Brain and Spinal Cord of Rats.

This study has examined the acute effects of taurine (TAU) and of its two immediate homologs aminomethanesulfonic acid (AMSA) and homotaurine (HTAU) on the oxidative stress that develops in the brain of rats as a result of type 2 diabetes mellitus. Male Sprague-Dawley rats, 220-225 g in weight, were divided into groups of 6 each, and treated with a single intraperitoneal (i.p.) dose of streptozotocin (STZ) in 10 mM citrate buffer pH 4.5 (60 mg/kg). The treatment compound (AMSA, HTAU or TAU) was administered by the i.p. route in two equal doses (1.2 mM/kg each) at 75 and 45 min before STZ. Control rats received only 10 mM citrate buffer pH 4.5 or only STZ by the i.p. route. The rats were sacrificed at 24 h after a dosing with STZ under general anesthesia, and their brains and spinal cords collected by the freeze clamp technique. A portion of brain, of a brain area (cerebellum, cortex, brain stem) or of spinal cord from each animal was extracted into 0.1 M PBS pH 7.4, and the extract was used for the assay of malondialdehyde (MDA), nitric oxide (NO), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD). An extract for the assay of the reduced (GSH) and disulfide (GGSG) forms of glutathione was prepared in similar manner but using 2% metaphosphoric acid plus 0.1 M PBS pH 8.0 as the extracting medium. Diabetes was found to markedly increase the formation of MDA (by 160-202%), NO (by 138-313%) and GSSG (by 103-241%), and to lower the values of GSH (by 57-65%), GSH/GSSG ratio (79-89%) and activities of CAT (by 61-69%), GPx (by 52-66%) and SOD (by 55-68%) in the brain, brain areas and spinal cord relative to corresponding control values (all at p < 0.001). These effects were reduced to values that were generally at least one-half of those seen in untreated diabetic rats, with TAU providing a greater attenuation of the formation of MDA and NO, an about similar action on the depletion of GSH, and a lower action on the decrease in the GSH/GSSG ratio caused by diabetes than either AMSA or HMTAU. In contrast AMSA and HMTAU were about equipotent with each other and more potent than TAU in preventing the loss of antioxidant enzyme activities associated with diabetes. In short, pretreating diabetic rats with AMSA, HMTAU or TAU is found to protect the brain against changes in biochemical parameters indicative of oxidative stress, with potency differences among the test compounds varying within a narrow range.

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.

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.

Comparison of taurine and pantoyltaurine as antioxidants in vitro and in the central nervous system of diabetic rats.

This study has comparatively evaluated the antiradical and antilipid peroxidizing actions of taurine (TAU) and its N-pantoyl analog pantoyltaurine (PTAU) in vitro, and has determined the extent to which these findings agree with the in vivo ability of these compounds to prevent changes in plasma glucose and in indices of oxidative stress in the plasma, brain and spinal cord induced by the diabetogen streptozotocin (STZ) in Sprague-Dawley rats. Using free radical-generating and oxidizing systems, PTAU was found more effective than TAU in scavenging DPPH, hydroxyl, peroxyl, and superoxide anion radicals and peroxynitrite, and in preventing lipid peroxidation of a brain homogenate by iron (III)-dopamine and the oxidation of dopamine by iron (III). On the other hand, when administered intraperitoneally (i.p.) at a 1.2mM/kg dose, 75min and 45min before a single i.p., 60mg/kg, dose of (STZ), TAU was about equipotent with PTAU in attenuating STZ-induced increases in glucose, malondialdehyde (MDA) and nitric oxide (NO), and the loss of reduced glutathione (GSH) in plasma collected at 24h post STZ. Moreover, the analysis of concurrently collected brain and spinal cords samples revealed that both TAU and PTAU were able to equally reverse the increases in MDA and NO concentrations and to effectively counteract the decrease in the GSH/GSSG ratio caused by STZ. Likewise, both compounds were very effective in preventing the losses of tissue catalase, glutathione peroxidase and superoxide dismutase activities. A comparison of the results for spinal cord and for brain parts such as the cerebellum, cortex and brain stem suggested the existence of regional differences in antioxidant potency between TAU and PTAU, especially in terms of antioxidant enzymes. In general, differences in antiradical and antioxidant potencies between TAU and PTAU derived from in vitro test are not reliable indicators of the antioxidant potencies these compounds may subsequently manifest in a living organism.

Protective action of taurine, given as a pretreatment or as a posttreatment, against endotoxin-induced acute lung inflammation in hamsters.

To assess the effect of taurine on lipopolysaccharide (LPS)-induced lung inflammation, oxidative stress and apoptosis, female Golden Syrian hamsters were intratracheally instilled with bacterial LPS (0.02 mg in phosphate buffered saline (PBS) pH 7.4), before or after a 3-day intraperitoneal treatment with a single dose of taurine (50 mg/kg/day in PBS pH 7.4), and bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected at 24 hr after the last treatment. In comparison to BALF samples from animals receiving only PBS pH 7.4, and serving as controls, those of LPS-stimulated animals exhibited a higher count of both total leukocytes and neutrophils and increased expression of tumor necrosis factor receptor 1. In comparison to lungs from control animals, those from LPS-treated animals showed increased cellular apoptosis, lipid peroxidation, decreased glutathione levels, altered activities of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase) and focal inflammation confined to the parenchyma. A treatment with taurine was found to significantly attenuate all these alterations, with the protection being, in all instances, greater when given before rather than after LPS. The present results suggest that taurine is endowed with antiinflammatory and antioxidant properties that are protective in the lung against the deleterious actions of Gram negative bacterial endotoxin.

Attenuating effect of taurine on lipopolysaccharide-induced acute lung injury in hamsters.

This study has evaluated the ability of the semiessential amino acid taurine to attenuate lipopolysaccharide (LPS)-induced lung inflammation, oxidative stress and apoptosis in a small animal model. For this purpose, bacterial LPS (0.02mg in phosphate buffered saline (PBS) pH 7.4) was instilled intratracheally into female Golden Syrian hamsters, before or after a 3-day intraperitoneal treatment with a single dose (50mg/kg in PBS pH 7.4) of taurine. At 24h after the last treatment, lung tissue and bronchoalveolar lavage fluid (BALF) samples were collected. In comparison to samples from animals receiving only PBS pH 7.4, serving as controls, those of LPS-stimulated animals exhibited a higher count of both total leukocytes and neutrophils in the BALF, and increased incidence of apoptosis, depletion of intracellular glutathione and evidence of inflammation confined to the parenchyma in the lung. In addition, LPS caused cells in the BALF to exhibit a higher expression of tumor necrosis factor-1, a higher activity of caspase-3, marked lipid peroxidation, and altered activities of catalase, glutathione peroxidase and superoxide dismutase relative to control samples. In contrast, a treatment with taurine was found to significantly attenuate all of the cellular and biochemical alterations induced by LPS, more so when given before rather than after the endotoxin. The present results suggest that taurine possesses intrinsic antiinflammatory and antioxidant properties that may be of benefit against the deleterious actions of LPS in the lung.