Chronic kidney disease: cardiac and renal angiotensin-converting enzyme (ACE) 2 expression in rats after subtotal nephrectomy and the effect of ACE inhibition.

Renin-angiotensin system blockade slows but does not prevent the cardiovascular complications of chronic kidney disease (CKD). Angiotensin-converting enzyme (ACE) 2 is differentially regulated in acute kidney injury, with increased cardiac ACE2 but decreased kidney ACE2 levels. This study investigated the effect of long-term ACE inhibition on cardiac and renal ACE2 in rats with CKD induced by subtotal nephrectomy (STNx). Sprague-Dawley rats had sham (control) or STNx surgery. Control rats received vehicle (n = 9) and STNx rats ramipril (1 mg kg(-1) day(-1); n = 10) or vehicle (n = 10) for 28 days. Subtotal nephrectomy resulted in impaired creatinine clearance (P < 0.05), proteinuria (P < 0.05), renal fibrosis (P < 0.05) and reduced renal cortical ACE2 mRNA (P < 0.05) and activity (P < 0.05). In rats with CKD, ramipril improved creatinine clearance (P < 0.05) and was associated with an increase in cortical but not medullary ACE2 activity (P < 0.05). Compared with control rats, STNx rats were hypertensive (P < 0.01), with increased left ventricular end-diastolic pressure (LVEDP; P < 0.01), left ventricular hypertrophy (LVH; P < 0.05) and interstitial (P < 0.05) and perivascular fibrosis (P < 0.01). In rats with CKD, ramipril decreased blood pressure (P < 0.001) and reduced LVEDP (P < 0.01), LVH (P < 0.01) and perivascular fibrosis (P < 0.05) but did not significantly reduce interstitial fibrosis. There was no change in cardiac ACE2 in rats with CKD compared with control rats. In rats with CKD, ACE inhibition had major benefits to reduce blood pressure and cardiac hypertrophy and to improve creatinine clearance, but did not significantly impact on cardiac ACE2, cardiac interstitial fibrosis, renal fibrosis or proteinuria. Thus, in rats with CKD, renal ACE2 deficiency and lack of activation of cardiac ACE2 may contribute to the progression of cardiac and renal tissue injury. As long-term ACE inhibition only partly ameliorated the adverse cardio-renal effects of CKD, adjunctive therapies that lead to further increases in ACE2 activity may be needed to combat the cardio-renal complications of CKD.

Angiotensin II mediates epithelial-to-mesenchymal transformation in tubular cells by ANG 1-7/MAS-1-dependent pathways.

Epithelial-to-mesenchymal transformation (EMT) of tubular cells into a myofibroblastic phenotype is an important mediator of renal scarring in chronic nephropathy. This study examines the role of the renin-angiotensin system (RAS) in this process. NRK-52E cells were exposed to angiotensin (ANG) II and ANG 1-7 in the presence or absence of inhibitors and agonists of RAS signaling. EMT was assessed at 3 days by expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin and the induction of a myofibroblastic phenotype. Expression of fibrogenic growth factors and matrix proteins was assessed by RT-PCR and immunofluorescence microscopy. To confirm findings in vivo, rats were also infused with ANG 1-7 (24 microg*kg(-1)*h(-1)) or saline via an osmotic minipump for 10 days, and renal fibrogenesis was then assessed. Treatment of NRK-52E cells with ANG II induced characteristic changes of EMT. Selective blockade of the AT(1) receptor or the AT(2) receptor failed to inhibit ANG II-induced EMT. However, blockade of the ANG 1-7 receptor, Mas-1, was able to prevent ANG II-dependent EMT. To confirm these findings, both ANG 1-7 and the selective Mas receptor agonist, AVE-0991, were able to induce NRK-52E cells in a dose-dependent manner. Exposing cells to recombinant ACE2 was also able to induce EMT. In addition, an infusion of ANG 1-7 induced the tubular expression of alpha-SMA and the expression of matrix proteins in the kidney. ANG II is a potent stimulus for EMT, but not through conventional pathways. This study points to the possible limitations of conventional RAS blockade, which not only fails to antagonize this pathway, but also may enhance it via augmenting the synthesis of ANG 1-7.

Metabolic effects of low dose angiotensin converting enzyme inhibitor in dietary obesity in the rat.

BACKGROUND AND AIMS: Given the recent observation of a local renin-angiotensin system (RAS) in adipose tissue, and its association with obesity-related hypertension, the metabolic effects of treatment with a low dose angiotensin converting enzyme inhibitor (ACEI) were investigated in a rodent model of diet-induced obesity. METHODS AND RESULTS: Male Sprague Dawley rats were exposed to either standard laboratory chow (12% calories as fat) or palatable high fat (30% calories as fat) diet for 12 weeks. A subset from both dietary groups was given low dose ACEI in drinking water (perindopril, 0.3 mg/kg/day) throughout the study. The high fat diet increased body weight, adiposity, circulating leptin and insulin and in the liver we observed fat accumulation and increased tissue ACE activity. Treatment with perindopril decreased food intake and circulating insulin in both diet groups, and hepatic ACE activity in high fat fed animals only. Decreased plasma leptin concentration with ACE inhibition was only evident in chow fed animals. These effects were independent of any blood pressure lowering effect of ACE inhibition. CONCLUSION: Chronic low dose ACEI treatment reduced circulating insulin and leptin levels with some reduction in food intake in chow fed rats. Fewer beneficial effects were observed in obesity, and further work is required to investigate higher ACEI doses. Our data suggest a reduction in hepatic ACE activity may affect lipid accumulation and other inflammatory responses, as well as improving insulin resistance. Our findings may have implications for maximizing the clinical benefit of ACEI in patients without overt cardiovascular complications.

Undernutrition during suckling in rats elevates plasma adiponectin and its receptor in skeletal muscle regardless of diet composition: a protective effect?

OBJECTIVE: Nutrition during critical periods in early life may increase the subsequent risk of obesity, hypertension and metabolic diseases in adulthood. Few studies have focused on the long-term consequences of poor nutrition during the suckling period on the susceptibility to developing obesity when exposed to a palatable cafeteria-style high-fat diet (CD) after weaning. DESIGN: This study examined the impact of early undernutrition, followed by CD exposure, on blood pressure, hormones and genes important for insulin sensitivity and metabolism and skeletal muscle mRNA expression of adiponectin receptor 1 (AdipoR1), carnitine palmitoyl-transferase I (CPT-1), cytochrome c oxidase 4 (COX4) and peroxisome proliferator-activated receptor alpha (PPARalpha). Following normal gestation, Sprague-Dawley rat litters were adjusted to 18 (undernourished) or 12 (control) pups. Rats were weaned (day 21) onto either palatable CD or standard chow. RESULTS: Early undernourished rats were significantly lighter than control by 17 days, persisting into adulthood only when animals were fed chow after weaning. Regardless of litter size, rats fed CD had doubled fat mass at 15 weeks of age, and significant elevations in plasma leptin, insulin and adiponectin. Importantly, undernutrition confined to the suckling period, elevated circulating adiponectin regardless of post-weaning diet. Blood pressure was reduced in early undernourished rats fed chow, and increased by CD. Early undernutrition was associated with long-term elevations in the expression of AdipoR1, CPT-1, COX4 and PPARalpha in skeletal muscle. CONCLUSION: This study demonstrates the important role of early nutrition on body weight and metabolism, suggesting early undernourishment enhances insulin sensitivity and fatty-acid oxidation. The long-term potential benefit of limiting nutrition in the early postnatal period warrants further investigation.

Acute kidney injury in the rat causes cardiac remodelling and increases angiotensin-converting enzyme 2 expression.

Patients with kidney failure are at high risk of a cardiac death and frequently develop left ventricular hypertrophy (LVH). The mechanisms involved in the cardiac structural changes that occur in kidney failure are yet to be fully delineated. Angiotensin-converting enzyme (ACE) 2 is a newly described enzyme that is expressed in the heart and plays an important role in cardiac function. This study assessed whether ACE2 plays a role in the cardiac remodelling that occurs in experimental acute kidney injury (AKI). Sprague-Dawley rats had sham (control) or subtotal nephrectomy surgery (STNx). Control rats received vehicle (n = 10), and STNx rats received the ACE inhibitor (ACEi) ramipril, 1 mg kg(-1) day(-1) (n = 15) or vehicle (n = 13) orally for 10 days after surgery. Rats with AKI had polyuria (P < 0.001), proteinuria (P < 0.001) and hypertension (P < 0.001). Cardiac structural changes were present and characterized by LVH (P < 0.001), fibrosis (P < 0.001) and increased cardiac brain natriuretic peptide (BNP) mRNA (P < 0.01). These changes occurred in association with a significant increase in cardiac ACE2 gene expression (P < 0.01) and ACE2 activity (P < 0.05). Ramipril decreased blood pressure (P < 0.001), LVH (P < 0.001), fibrosis (P < 0.01) and BNP mRNA (P < 0.01). These changes occurred in association with inhibition of cardiac ACE (P < 0.05) and a reduction in cardiac ACE2 activity (P < 0.01). These data suggest that AKI, even at 10 days, promotes cardiac injury that is characterized by hypertrophy, fibrosis and increased cardiac ACE2. Angiotensin-converting enzyme 2, by promoting the production of the antifibrotic peptide angiotensin(1-7), may have a cardioprotective role in AKI, particularly since amelioration of adverse cardiac effects with ACE inhibition was associated with normalization of cardiac ACE2 activity.

Angiotensin converting enzyme 2 (ACE2) activity in fetal calf serum: implications for cell culture research.

Cell culture experiments often employ the use of culture media that contain fetal calf serum (FCS). The angiotensin peptides angiotensin II and angiotensin 1-7 have opposing effects with angiotensin converting enzyme 2 (ACE2) being the enzyme predominantly responsible for generating angiotensin 1-7 from angiotensin II. The effect of FCS on angiotensin peptides has not previously been described. We have shown that FCS has ACE2 enzyme activity capable of degrading angiotensin II and generating angiotensin 1-7. Researchers should be aware that FCS possesses ACE2 activity and that heat-treating FCS to 56 degrees C only partially inhibits this enzyme activity, whereas heat-treating to 70 degrees C completely abolishes ACE2 activity.

Leptin reduces food intake but does not alter weight regain following food deprivation in the rat.

OBJECTIVE: When animals are allowed free access to food following an extended period of food restriction, body weight is steadily restored to the pre-food restriction level, ie to a specific body weight 'set-point'. We tested the proposition that leptin is used as a signal by the brain to regulate body weight 'set-point'. To this end, we determined whether long-term leptin infusion in rats would prevent the normal weight regain after food restriction. METHODS: Male Sprague-Dawley rats received leptin (leptin-treated) or saline (vehicle-treated) by intravenous infusion. After a 2 week run-in period, food intake was adjusted to 50% of each individual's normal intake for 12 days. Two days prior to the return of unlimited access to food, one group of animals received continuous leptin infusion at 3 micro g/h for the next 14 days. Blood samples taken from the tail vein were used to determine leptin concentrations. A third group of animals that did not undergo food restriction but received saline infusion served as control. As leptin acts in the brain to modulate neuropeptide Y (NPY) levels, hypothalamic NPY content was measured at the end of the study. RESULTS: Food restriction to 50% normal intake for 12 days induced a 20% weight loss and significant reductions in plasma leptin compared with non-restricted control rats (0.5+/-0.1 vs 2.6+/-0.4 ng/ml, P<0.05). Intravenous infusion of leptin increased leptin concentrations four-fold compared with vehicle-treated animals (9.5+/-1.3 vs 2.2+/-0.4 ng/ml, P<0.05). The infusion of leptin attenuated the increase in daily food intake after free access to food was resumed (P<0.05 at 4, 6 and 8 days). Despite this, both groups of previously restricted rats had regained the same amount of weight after 12 days of ad libitum feeding. No difference was noted in NPY levels measured in the arcuate nucleus and the paraventricular nucleus, in line with the similar amounts of food eaten by all rats at the end of the experiment. CONCLUSION: Increasing plasma leptin concentrations just prior to the end of a period of food restriction reduced subsequent food intake, but did not appear to exert a major influence on the body weight 'set-point', as leptin did not prevent weight regain. The results of the present study suggest that leptin may be of little value in maintaining weight loss in individuals who have lost weight through dieting. Further research is required to understand the role of leptin in the regulation of energy balance.