Aika Hagiwara

Improvement of Physical Decline Through Combined Effects of Muscle Enhancement and Mitochondrial Activation by a Gastric Hormone Ghrelin in Male 5/6Nx CKD Model Mice

Because a physical decline correlates with an increased risk of a wide range of disease and morbidity, an improvement of physical performance is expected to bring significant clinical benefits. The primary cause of physical decline in 5/6 nephrectomized (5/6Nx) chronic kidney disease model mice has been regarded as a decrease in muscle mass; however, our recent study showed that a decrease in muscle mitochondria plays a critical role. In the present study, we examined the effects of a gastric hormone ghrelin, which has been reported to promote muscle mitochondrial oxidation, on the physical decline in the chronic kidney disease model mice, focusing on the epigenetic modulations of a mitochondrial activator gene, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Ghrelin treatment improved a decline in exercise endurance of 5/6Nx mice, associated with an increase in both of the muscle mass and mitochondrial amount. The expression level of PGC-1α was decreased in the skeletal muscle of 5/6Nx mice, which was associated with an increase in the methylation ratio of the cytosine residue at 260 base pairs upstream of the initiation point. Conversely, ghrelin treatment de-methylated the cytosine residue and increased the expression of PGC-1α. A representative muscle anabolic factor, IGF-1, did not affect the expression of PGC-1α and muscle mitochondrial amount, although it increased muscle mass. As a result, IGF-1 treatment in 5/6Nx mice did not increase the decreased exercise endurance as effectively as ghrelin treatment did. These findings indicate an advantage of ghrelin treatment for a recovery of physical decline.

Treatment of sarcopenia and glucose intolerance through mitochondrial activation by 5-aminolevulinic acid

Recently, sarcopenia has attracted attention as therapeutic target because it constitutes a risk factor for metabolic and cardiovascular diseases. We focused 5-aminolevulinic acid (ALA) which act as electron carriers in the mitochondrial electron transport system. The mice that received ALA for 8 weeks gained muscle strength and endurance, and exhibited increased muscle mass and mitochondrial amount. Administration of ALA to sarcopenia mice aged 100 weeks and chronic kidney disease (CKD) model mice also increased muscle mass and improved physical performance. Metabolome analysis revealed increased branched-chain amino acids (BCAAs) levels in the skeletal muscle of ALA-treated mice. Quantitative PCR analysis revealed decreased expression levels in branched-chain amino acid transaminases (BCATs) that degrade BCAAs and other muscle-degrading factors, and increased levels of mitochondria-activating factors. We also studied in cultured myocytes and obtained compatible results. ALA-treated mice tended to increase body weight, but reduced blood glucose level. These suggested that ALA treatment not only activated muscle mitochondria but also enhanced muscle mass through an increase in BCAAs contents, as to improve muscle strength, endurance and glucose tolerance in mice. In these ways, muscle mitochondrial activation with ALA is suggested to be useful for the treatment of sarcopenia and glucose intolerance.

Ghrelin treatment improves physical decline in sarcopenia model mice through muscular enhancement and mitochondrial activation

Chronic kidney disease (CKD) impairs physical performance in humans, which leads to a risk of all-cause mortality. In our previous study, we demonstrated that a reduction in muscle mitochondria rather than muscle mass was a major cause of physical decline in 5/6 nephrectomized CKD model mice. Because ghrelin administration has been reported to enhance oxygen utilization in skeletal muscle, we examined the usefulness of ghrelin for a recovery of physical decline in 5/6 nephrectomized C57Bl/6 mice, focusing on the epigenetic modification of peroxisome proliferator activated receptor gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. The mice were intraperitoneally administered acylated ghrelin (0.1 nmol/gBW; three times per week) for a month. Muscle strength and exercise endurance were measured by using a dynamometer and treadmill, respectively. Mitochondrial DNA copy number was determined by quantitative PCR. The methylation levels of the cytosine residue at 260 base pairs upstream of the translation initiation point (C-260) of PGC-1α, which has been demonstrated to decrease the expression, was evaluated by methylation-specific PCR and bisulfite genomic sequencing methods after the ghrelin administration. Ghrelin administration improved both muscle strength and exercise endurance in the mice and was associated with an increase in muscle mass and muscle mitochondrial content. Ghrelin administration decreased the methylation ratio of C-260 of PGC-1α in the skeletal muscle and increased the expression. Therefore, ghrelin administration effectively reduced the physical decline in 5/6 nephrectomized mice and was accompanied with an increased mitochondrial content through de-methylation of the promoter region of PGC-1α in the muscle.

TRANSIENT SALT LOADING CAUSES PERSISTENT HYPERTENSION THROUGH EPIGENETIC MODIFICATION OF THE RENAL ARTERIOLES

Objective: We have previously reported that the medial hypertrophy of the renal arterioles induced by transient salt loading causes sustained elevation of blood pressure in spontaneous hypertensive rat (SHR) even after the salt loading had completed [Oguchi et al. Hypertension 2014]. Epigenetic modification of gene expression has attracted attention as a possible mechanism for sustained biological effects and onset of hypertension. The present study investigated the significance of histone acetylation in each segment of the kidney in the induction of hypertension after transient salt loading. Design and method: C57bl6 mice were implanted deoxycorticosterone acetate (DOCA) pellets and given drinking water containing 1% NaCl for 2 weeks to induce hypertension. We evaluated blood pressure, histological findings and gene expressions of the kidney during and after the transient salt loading. The degree of histone acetylation was assessed by immunostaining of acetylated H3 and H4 in each segment of the kidney including renal arterioles, segmental arteries, glomeruli and tubules. Gene expressions were examined in each segment of the kidney collected by laser capture microdissection (LCM). Results: Transient salt loading caused elevation in blood pressure during and even after stopping salt loading associated with persistent medial hypertrophy of renal arterioles. In the media of renal arterioles, histone acetylation was enhanced during salt loading, and the enhanced histone acetylation persisted even after stopping salt loading. The gene expression of MMPs in the renal arterioles collected by LCM increased during salt loading, and did not decline even after stopping salt loading. On the other hand, in the segmental arteries, neither hyper-acetylation nor hyper-expression of MMPs was observed. Also, in the tubules, enhanced histone acetylation by the salt loading returned to the initial level after the completion. Conclusions: The persistent medial hypertrophy along with focally sustained histone hyper-acetylation and elevation in MMPs expressions in the renal arterioles were suggested to cause sustained elevation of blood pressure after transient salt loading. The focal epigenetic modification in the kidney due to environmental factors would participate in the onset of persistent hypertension.

A MASS IMAGING TECHNIQUE REVEALED A RENO-PROTECTIVE EFFECT OF THE XANTHINE OXIDASE INHIBITOR FEBUXOSTAT IN THE ISCHEMIC KIDNEY BY PROMOTING ATP RECOVERY IN THE CORTEX

Objective: The kidney has different energy metabolism depending on the region. However, the distribution of phosphorylated adenosine (ATP, ADP and AMP) and their alteration after transient ischemia have not been known due to the technical difficulties. Design and method: Imaging mass spectrometry (IMS) with metabolome analysis is a novel technique to quantify the small metabolites in the tissues. We performed the IMS analysis in the ischemic kidney after transient ischemia by renal artery clipping. Results: In the normal kidney, ATP was significantly rich in both the cortex and outer medulla. After transient ischemia, ATP in the cortex degraded and the energy charge value decreased within a minute. ATP in the inner medulla did not decrease within a minute and needed 10 minutes to start decreasing. After the 10 minutes of ischemia, total adenylates decreased in the cortex, although the decrease in energy charge value was homogeneous in the kidney. During the 24 hours reperfusion after 10 minutes ischemia, restoration of total adenylates in the cortex was not sufficient. Febuxostat is a xanthine oxidase inhibitor which might promote reuse of hypoxanthine as a progenitor of adenylates and therefore might improve the restoration of total adenylates and ATP after transient ischemia. The administration of febuxostat in accordance with the reperfusion period supported the restoration of ATP level in the cortex and improved renal function which was impaired by transient ischemia. Conclusions: In these ways, IMS revealed the region-specific alteration of phosphorylated adenosine the ischemic kidney and the novel effect of febuxostat on the restoration of total adenylates and ATP in the cortex after transient ischemia.

MPS 07-01 THE ROLE OF HISTONE ACETYLATION AND MMP EXPRESSION IN VASCULAR REMODELING OF RENAL ARTERIOLES IN DOCA-SALT HYPERTENSIVE MICE

Objective: The role of salt intake in the development of hypertension is familiar, but the mechanism has not been revealed. Our previous report showed that the medial hypertrophy of renal arterioles induced by transient salt loading caused lasting renin elevation resulting in elevation of blood pressure in spontaneous hypertensive rat. The present study investigated the significance of epigenetic modulation of renal arterioles in the induction of medial hypertrophy after transient salt loading. Design and Method: Male 8 week old C57bl6 mice were implanted deoxycorticosterone acetate (DOCA) pellets and given drinking water containing 1% NaCl for 2 weeks to introduce salt-induced hypertension. Blood pressure was measured by tail-cuff method during and after transient salt loading. Histological examinations of the kidney were performed. Gene expressions in the whole kidney and renal arterioles were quantified using real time PCR and laser capture microdissection. To investigate the significance of histone acetylation, DOCA-salt mice were treated with histone acetyltransferase (HAT) inhibitor curcumin during salt loading. Results: Transient salt loading caused medial hypertrophy of renal arterioles and elevation in blood pressure during and even after salt loading. Real time PCR in the whole kidney revealed that the gene expressions of HATs such as CBP and p300 were elevated; and conversely those of histone deacetylases such as Sirts and HDACs were decreased during salt loading. Histone H3 and H4 acetylation in the promoter region of MMP-2 gene was enhanced during salt loading. MMP-2 and p300 in the renal arterioles were also increased during salt loading. Curcumin reduced the medial hypertrophy of renal arterioles and elevation in blood pressure partially. Conclusions: The lasting medial hypertrophy after transient salt loading was suggested to be caused by the increase of MMP-2 expression along with histone acetylation. Increased expressions of HATs and decreased expressions of HDACs were supposed to be involved in the enhancement of histone acetylation.

PS 10-01 SIGNIFICANCE OF RENAL AUTONOMIC NERVES FOR BODY WEIGHT REDUCTION-BY TOFOGLIFLOZIN

Objective: Sodium-glucose co-transporter (SGLT) 2 inhibitors decrease not only serum glucose level but also the body weight significantly. However, the mechanism responsible for the weight reduction has not been elucidated. Renal denervation (RDN) is also reported to affect the body weight. Therefore, we speculated that renal autonomic nerves might be involved in the mechanism by which SGLT2 inhibitors reduces the body weight. The present study examined whether RDN affects the weight reduction by a SGLT2 inhibitor, by using diet-induced obese mice treated with a SGLT2 inhibitor, tofogliflozin (tofo). Design and Method: The C57bl/6 mice fed on a high-fat diet were divided into 4 groups; control, RDN, SGLT2 inhibitor (tofo) and tofo with RDN groups. We measured the body weight, the systolic blood pressure and the glucose tolerance. The SGLT2 inhibitor was administered to mice from 8 to 16 weeks old by mixing it in the high-fat diet (tofogliflozin 5 mg/g pellet, 60 Kcal% fat). RDN was performed at 7 weeks old by surgically stripping renal arteries and coating the vessels with a solution of 10% phenol in ethanol. Results: The body weight at 16 weeks old significantly decreased in tofo group. In that group, catecholamine levels and activity of hormone sensitive lipase (HSL) in the adipose tissue were increased. On the other hand, RDN significantly weakened the degree of body weight reduction by a treatment with tofogliflozin associated with suppression of the catecholamine levels and HSL activity. In RDN without tofo group, the body weight slightly decreased; however, the HSL activity was not changed. Conclusions: These results indicated that HSL activation in the adipose tissue by tofogliflozin were affected by the activity of renal autonomic nerves. The renal autonomic nerves would have a role in the control of body weight.

MPS 07-06 5-AMINOLEVULINIC ACID, A MATERIAL FOR MITOCHONDRIAL ELECTRON CARRIER, IMPROVES EXERCISE ENDURANCE THROUGH BOTH MUSCLE ENHANCEMENT AND ACTIVATION OF MUSCLE MITOCHONDRIA IN SARCOPENIC MICE.

Objective: Sarcopenia affects not only physical performance, but also increases the risk for cardiovascular diseases. Aminolevulinic acid (ALA) is a raw material for the mitochondrial electron carriers, heme and cytochrome C. In the present study, physical performance of ALA-treated mice (0.03%-ALA/ chow weight) was examined as to elucidate the usefulness of ALA for the treatment of sarcopenia. Design and Method: Alteration in physical performance and muscle mass were examined in ALA-treated mice. Harvested skeletal muscle was examined for expression of genes related to mitochondrial amount and function. Results: Muscle mass and mitochondrial amount were increased in the skeletal muscle of ALA-treated mice. Expressions of the mitochondrial activation genes, PGC1a and UCP3 were up-regulated. Decreased muscle strength, exercise endurance and glucose tolerance of aging mice and 5/6 nephrectomized mice, which were sarcopenic with reduced amount of muscle mitochondria, were improved by the ALA-feeding for 8weeks. In the experiments using cultured myocytes treated with ALA, expressions of PGC1a and UCP3, and mitochondrial amount were increased, associated with augmented oxygen utilization. Suppression of electron transport by antimycin A attenuated the increases in oxygen utilization, PGC1a expression and mitochondrial amount in ALA-treated myocytes. Conclusions: ALA improved exercise endurance and glucose intolerance of sarcopenic mice through increases in muscle mass, muscle PGC1a expression and mitochondrial amount. These results indicated the usefulness of ALA for improvement of physical performance and glucose tolerance in sarcopenic patients through both muscle enhancement and activation of muscle mitochondria.

MPS 11-04 Elucidation of tubulo-interstitial injury in chronic kidney ischemia by use of novel renal artery coiling model

Objective: Recent studies emphasize an essential role for tubulointerstitial hypo-perfusion in the progression of chronic kidney disease (CKD) as a common pathway to end stage renal disease (ESRD). However, most popular animal models which aimed to simulate renal ischemia have been ischemic-reperfusion injury model, which shows major characteristics of acute kidney injury. Thus, a novel method is needed to elucidate the real mechanism of renal injury caused by chronic interstitial hypo-perfusion. Design and Method: We developed a novel renal ischemia model by setting a titanium coil around a left renal artery of right hemi-nephrectomized mice. The renal blood flow was reduced for 80% by setting a coil. The inside diameter of coil is adjusted from 0.17 to 0.28 mm. The phenotype of this model was evaluated by renal function, histological changes and real-time quantitative PCR. Results: After transient decline in renal function until post-operative day 14, slowly progression of renal dysfunction and elevation of N-acetyl-&bgr;-D-glucosaminidase (NAG) in urine were observed in the chronic renal ischemia model from post-operative day 14 to 84. A histological observation of the ischemic kidney on post-operative day 7 revealed no remarkable changes from control kidneys and there are no findings of tissue injury and infiltration of inflammatory cells. However, the progression of tubulointerstitial fibrosis and the increase in the markers for apoptosis and oxidative stress were significant from post-operative day 14 to 84. Real-time quantitative PCR analysis in ischemic kidney revealed the progressive changes of the hypoxia-responsive gene expressions. Conclusions: These data suggests that the novel chronic renal ischemia model by coiling renal artery is an appropriate animal model for CKD, which shows a slowly progressive tubulointerstitial injury.

PS 16-03 EFFECTS OF 72 HOURS FASTING AND REFEEDING ON FAT OXIDATION AND WEIGHT SUPPRESSION

Objective: Intermittent fasting has been shown to have several health benefits including reduced blood pressure, increased insulin sensitivity, stress resistance and reduced morbidity. However, the mechanism for the beneficial effects has not been well investigated. Design and method: Body weight and body temperature of c57BL/6 mice were examined when they were maintained on the intermittent fasting (IF) of 72 hours fasting and 96 hours refeeding in a week for 4 weeks, and subsequently administered a high fat diet (HFD) for 4 weeks. After the IF and HFD administration, glucose tolerance test and respiratory gas analysis were performed. Expressions of genes responsible for fat oxidation and lipolysis in the skeletal muscle and white adipose tissue were examined by PCR analysis. Results: There was no significant difference in body weight and body temperature between the adlib eating and IF groups before HFD administration. After HFD administration, the body weight in IF group was significantly lower and the body temperature became higher when compared to adlib eating group. Glucose tolerance test after HFD showed a decrease in glucose level in IF group. In the PCR analysis after HFD, expressions of genes responsible for fat oxidation, heat production and lipolysis (UCP1, DiO2, AGTL and HSL) were increased in the skeletal muscle and white adipose tissue. Expressions of hormone receptors responsible for fat oxidation (GHSR and AdipoR1) were also increased. Conclusions: The operation of IF resulted in suppression of body weight gain after HFD administration. From the results of PCR analysis, promotions of fat oxidation, lipolysis and heat production in the skeletal muscle and white adipose tissue in IF group were suggested to be resulted in suppression of body weight gain. Furthermore, the alteration in hormone responses might be involved in these beneficial effects of the IF.