Elisa Mieko Suemitsu Higa

Increase in kinins on post-exercise hypotension in normotensive and hypertensive volunteers.

Abstract Post-exercise hypotension is an important event for blood pressure regulation, especially in hypertensive individuals. Although post-exercise hypotension is a well-known phenomenon, the mechanism responsible is still unclear. The kallikrein-kinin system is involved in blood pressure control, but its role in post-exercise hypotension has not yet been investigated. Thus, the purpose of this study was to investigate the involvement of the vasodilators bradykinin and des-Arg9-BK and kallikrein activity in post-exercise hypotension promoted by 35 min of cycle ergometer (CE) or circuit weight-training (CWT) bouts in normotensive and hypertensive individuals. A significant decrease in mean arterial pressure at 45 and 60 min after CE and 45 min after CWT was observed in normotensive individuals. Hypertensive values of mean arterial pressure were significantly reduced at 45 and 60 min after CE and at 60 min after CWT. Before exercise, plasma bradykinin concentrations and kallikrein activity were higher in hypertensive compared to normotensive volunteers. Kinin levels increased in the groups evaluated at the end of the training period and 60 min post-exercise. These data suggest that the kallikrein-kinin system may be involved in post-exercise hypotension in normotensive and hypertensive individuals subjected to CE and CWT bouts.

Chronic conventional resistance exercise reduces blood pressure in stage 1 hypertensive men.

Abstract Moraes, MR, Bacurau, RFP, Casarini, DE, Jara, ZP, Ronchi, FA, Almeida, SS, Higa, EMS, Pudo, MA, Rosa, TS, Haro, AS, Barros, CC, Pesquero, JB, Würtele, M, and Araujo, RC. Chronic conventional resistance exercise reduces blood pressure in stage 1 hypertensive men. J Strength Cond Res 26(4): 1122–1129, 2012—To investigate the antihypertensive effects of conventional resistance exercise (RE) on the blood pressure (BP) of hypertensive subjects, 15 middle-aged (46 ± 3 years) hypertensive volunteers, deprived of antihypertensive medication (reaching 153 ± 6/93 ± 2 mm Hg systolic/diastolic BP after a 6-week medication washout period) were submitted to a 12-week conventional RE training program (3 sets of 12 repetitions at 60% 1 repetition maximum, 3 times a week on nonconsecutive days). Blood pressure was measured in all phases of the study (washout, training, detraining). Additionally, the plasma levels of several vasodilators or vasoconstrictors that potentially could be involved with the effects of RE on BP were evaluated pre- and posttraining. Conventional RE significantly reduced systolic, diastolic, and mean BP, respectively, by an average of 16 (p < 0.001), 12 (p < 0.01), and 13 mm Hg (p < 0.01) to prehypertensive values. There were no significant changes of vasoactive factors from the kallikrein-kinin or renin-angiotensin systems. After the RE training program, the BP values remained stable during a 4-week detraining period. Taken together, this study shows for the first time that conventional moderate-intensity RE alone is able to reduce the BP of stage 1 hypertensive subjects free of antihypertensive medication. Moreover, the benefits of BP reduction achieved with RE training remained unchanged for up to 4 weeks without exercise.

Nitric oxide (NO) is associated with gentamicin (GENTA) nephrotoxicity and the renal function recovery after suspension of GENTA treatment in rats.

GENTA nephrotoxicity is likely to be caused, among other factors, by an increase of vasoconstrictors or a decrease of vasodilators such as NO. Few days after discontinuing GENTA treatment, the renal function is recovered, but if risk factors like advanced age, previous renal dysfunction, simultaneous use of other nephrotoxic drugs or dehydration are present, severe and progressive renal disease occurs. The aim of this study was to evaluate the renal function in rats during GENTA treatment and after its suspension as well as its relationship with NO. Rats were treated with water (vehicle, CTL) or GENTA (100 mg/kg BW) intraperitonially during 10 days; both n=24. Twelve animals of each group were sacrificed after blood and 24 h urine were collected, and their kidneys were removed for histology. In another rats this procedure underwent after 20 or 30 days of GENTA suspension. GENTA treated group developed a marked decrease in renal function, characterized by an increased serum urea and decreased creatinine clearance; NO was increased in the serum and decreased in the urine; all P < 0.01 vs CTL. Acute tubular necrosis was confirmed in GENTA treated group. After GENTA suspension we observed a normalization of urea, creatinine clearance and serum and urinary NO; the histological lesions were also attenuated. We suggest that NO could play a role in GENTA induced nephrotoxicity and recovery. The understanding of this physiopathology could be an useful tool to prevent or blunt the nephrotoxicity progression, mainly when risk factors are present.

Kefir administration reduced progression of renal injury in STZ-diabetic rats by lowering oxidative stress

This study aimed at assessing the effects of Kefir, a probiotic fermented milk, on oxidative stress in diabetic animals. The induction of diabetes was achieved in adult male Wistar rats using streptozotocin (STZ). The animals were distributed into four groups as follows: control (CTL); control Kefir (CTLK); diabetic (DM) and diabetic Kefir (DMK). Starting on the 5th day of diabetes, Kefir was administered by daily gavage at a dose of 1.8 mL/day for 8 weeks. Before and after Kefir treatment, the rats were placed in individual metabolic cages to obtain blood and urine samples to evaluate urea, creatinine, proteinuria, nitric oxide (NO), thiobarbituric acid reactive substances (TBARS) and C-reactive protein (CRP). After sacrificing the animals, the renal cortex was removed for histology, oxidative stress and NOS evaluation. When compared to CTL rats, DM rats showed increased levels of glycemia, plasmatic urea, proteinuria, renal NO, superoxide anion, TBARS, and plasmatic CRP; also demonstrated a reduction in urinary urea, creatinine, and NO. However, DMK rats showed a significant improvement in most of these parameters. Despite the lack of differences observed in the expression of endothelial NO synthase (eNOS), the expression of inducible NO synthase (iNOS) was significantly lower in the DMK group when compared to DM rats, as assessed by Western blot analysis. Moreover, the DMK group presented a significant reduction of glycogen accumulation within the renal tubules when compared to the DM group. These results indicate that Kefir treatment may contribute to better control of glycemia and oxidative stress, which is associated with the amelioration of renal function, suggesting its use as a non-pharmacological adjuvant to delay the progression of diabetic complications.

Nitric Oxide‐Induced Murine Hematopoietic Stem Cell Fate Involves Multiple Signaling Proteins, Gene Expression, and Redox Modulation

There are a growing number of reports showing the influence of redox modulation in cellular signaling. Although the regulation of hematopoiesis by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been described, their direct participation in the differentiation of hematopoietic stem cells (HSCs) remains unclear. In this work, the direct role of nitric oxide (NO•), a RNS, in the modulation of hematopoiesis was investigated using two sources of NO•, one produced by endothelial cells stimulated with carbachol in vitro and another using the NO•‐donor S‐nitroso‐N‐acetyl‐d,l‐penicillamine (SNAP) in vivo. Two main NO• effects were observed: proliferation of HSCs—especially of the short‐term HSCs—and its commitment and terminal differentiation to the myeloid lineage. NO•‐induced proliferation was characterized by the increase in the number of cycling HSCs and hematopoietic progenitor cells positive to BrdU and Ki‐67, upregulation of Notch‐1, Cx43, PECAM‐1, CaR, ERK1/2, Akt, p38, PKC, and c‐Myc. NO•‐induced HSCs differentiation was characterized by the increase in granulocytic‐macrophage progenitors, granulocyte–macrophage colony forming units, mature myeloid cells, upregulation of PU.1, and C/EBPα genes concomitantly to the downregulation of GATA‐3 and Ikz‐3 genes, activation of Stat5 and downregulation of the other analyzed proteins mentioned above. Also, redox status modulation differed between proliferation and differentiation responses, which is likely associated with the transition of the proliferative to differentiation status. Our findings provide evidence of the role of NO• in inducing HSCs proliferation and myeloid differentiation involving multiple signaling. Stem Cells 2014;32:2949–2960

Electroacupuncture and Moxibustion Decrease Renal Sympathetic Nerve Activity and Retard Progression of Renal Disease in Rats

Background/Aim: Chronic kidney disease (CKD) is an increasing major public health problem worldwide. The sympathetic nervous system and nitric oxide play an important role in the pathogenesis of CKD. Traditional Chinese medicine has accumulated thousands of years of therapeutic experiences. Electroacupuncture (EA) and moxibustion (MO) are two such therapeutic strategies. The aim of this study was to investigate the renal and hemodynamic effects of EA-MO in an experimental model of a CKD. Methods: Male Wistar rats submitted to 5/6th nephrectomy (5/6 NX) were studied for 8 weeks. There were four groups: (1) control, normal rats; (2) NX, 5/6 NX only; (3) NX-AS, 5/6 NX and EA-MO session using sham points, and (4) NX-AM, 5/6 NX and EA-MO session using real acupoints. Biochemical and blood pressure studies, renal sympathetic nerve activity measurements, nitric oxide levels and the histopathological indices were assessed. Results: The EA- and MO-treated group presented significant improvement in all measured functional and histopathological parameters. Conclusion: These findings suggest that EA-MO had beneficial effects on CKD. This effect was probably achieved by the modulation of the renal sympathetic nerve activity and nitric oxide levels, leading to decreased blood pressure, which is associated with less proteinuria.

EFFECT OF CYCLOSPORIN A ON NITRIC OXIDE PRODUCTION IN CULTURED LLC-PK1 CELLS

The effect of Cyclosporin A on nitric oxide production was studied in cultured LLC- PK1 cells. For this purpose the cells were incubated with vehicle (olive oil, 10 μg/ml in DMSO), Cyclosporin A (CsA, 10 μg/ml), tumor necrosis factor (TNF-α, 150 U/ml) + interferon (IFN-γ, 500 U/ml) to upregulate NOS synthesis, and therefore NO production (used as a positive control), or CsA + TNF-α + IFN-γ. After 72 hours the culture medium was collected and nitrite was determined by the Griess method. The results were normalized to the protein harvested from these cells as measured by the Lowry method. Viability was determined by the exclusion of the fluorescent dyes (acridine orange and ethidium bromide). Intracellular calcium was measured spectrophotometrically using the fluorescent calcium indicator fura-2 AM. In CsA treated cells, the nitrite (pmoles/mg of protein) was decreased when compared to control (12.8 ± 0.5 vs. 18.3 ± 0.6; p < 0.05; both n = 8). TNF-α + IFN-γ increased the nitrite synthesis (52.0 ± 0.2; p < 0.05 vs. control; n = 6). This effect was decreased significantly by the simultaneous treatment with CsA (38.8 ± 0.3; p <0.05; n = 6). Cell viability in CsA group was decreased when compared to the control (84.7 ± 0.2% vs. 93.6 ± 0.1%; p < 0.05; both n = 10). TNF-α + IFN-γ had no effect on viability (93.0 ± 0.3%; n = 10). However, when combined with CsA, viability was decreased relative to the control (85.0 ± 0.2%; p < 0.05; n = 10). Acute (1 h) or chronic (72 h) treatment of LLC- PK1 cells with CsA had no effect on basal calcium levels. Our results demonstrate a reduced level of nitric oxide production in LLC- PK1 cells treated with CsA. There was no effect of the drug on intracellular calcium levels, however CsA treatment did reduce cellular viability. We suggest that, in part, the decreased levels of NO production are a secondary consequence of direct cell damage. However, CsA may also be exerting direct effects on NO synthesis through its interactions with both iNOS and cNOS. These results also provide a dual mechanism of action for CsA induced nephrotoxicity, that is, direct cell damage and interference with the NO system within the nephron.

Ascorbic acid reduces gentamicin-induced nephrotoxicity in rats through the control of reactive oxygen species

BACKGROUND & AIM Oxidative stress has been implicated in the pathophysiology of many forms of acute renal failure. The aim was examine the effect of vitamin C on oxidative stress and its relationship with nitric oxide on gentamicin-induced nephrotoxicity in rats. METHODS We utilized 32 Wistar rats allocated in four groups of eight animals each: control (CTL), vitamin C (VIT C), gentamicin (GENTA), and GENTA + VIT C; all groups were treated during seven days. RESULTS Serum urea and creatinine, serum and renal tissue malondialdehyde, blood superoxide anion and hydrogen peroxide in GENTA were increased vs CTL and vs VIT C, and decreased in GENTA + VIT C vs GENTA (all P < 0.05). Serum nitric oxide increased in GENTA vs CTL and vs VIT C, and reduced in GENTA + VIT C vs GENTA (P < 0.001). Urinary nitric oxide was reduced in GENTA vs CTL and vs VIT C and increased in GENTA + VIT C vs GENTA (P < 0.001). Severe degeneration of proximal tubules was present in GENTA, but only mild lesions were observed in GENTA + VIT C. CONCLUSION This study suggests that VIT C is a valuable tool to protect against GENTA-induced nephrotoxicity, by reducing reactive oxygen species and increasing the nitric oxide.

Neuroglobin is up-regulated in the cerebellum of pups exposed to maternal epileptic seizures

To evaluate a potential insult in the cerebellum of pups exposed to maternal epileptic seizures during intrauterine life, female rats were subjected to pilocarpine‐induced epilepsy. Pups from different litters were sacrificed at 1, 3, 7 and 14 post‐natal days (PN) and neuroglobin (Ngb) and gliosis were analyzed in the cerebellum by Western blotting (WB) and RT‐PCR. 14C‐l‐leucine‐[14C‐Leu] incorporation was used to analyze protein synthesis at PN1. Nitric Oxide (NO) and thiobarbituric acid‐reactive substances (TBARS) levels were also measured. Pups from naive mothers were used as controls. The mRNA level of Ngb was increased in experimental animals at PN1 (**p ≤ 0.001) and PN3 (**p ≤ 0.001), at PN7 (***p ≤ 0.0001) and at PN14 (**p ≤ 0.001) compared to the respective controls. The protein level of Ngb increased significantly in the experimental pups at PN1 (*p ≤ 0.05) and at PN3 (**p ≤ 0.001), when compared to the control pups at PN1 and PN3. At PN7 and PN14 no difference was found. The mRNA level of GFAP increased significantly about two times at PN3 (*p ≤ 0.05) and PN7 (*p ≤ 0.05) in the experimental pups when compared to the respective controls, but was unchanged in the other studied ages. Data showed that experimental pups at PN1 exhibited reduced (about 2 times, *p ≤ 0.05) total protein synthesis in the cerebellum when compared to control. No differences were found in the NO and TBARS levels. Our data support the hypothesis that an up‐regulation of Ngb could be a compensatory mechanism in response to the hypoxic–ischemic insults caused by seizures in pups during intrauterine life.

Endothelial nitric oxide synthase uncoupling as a key mediator of melanocyte malignant transformation associated with sustained stress conditions

Melanoma cell lines and cells corresponding to premalignant melanocytes were established by our group after subjecting a nontumorigenic murine melanocyte lineage, melan-a, to sequential cycles of anchorage blockade. Previous results showed that in melan-a cells the superoxide level increases after such procedure. Superoxide production during melanocyte de-adhesion was inhibited by L-sepiapterin, the precursor of eNOS cofactor BH4, and increased by the inhibitor of BH4 synthesis, DAHP, hence indicating a partial uncoupling state of eNOS. The eNOS uncoupling seems to be maintained in cells derived from melan-a, because they present decreased nitric oxide and increased superoxide levels. The inhibition of superoxide production in Tm5 melanoma cells with L-sepiapterin reinforces their eNOS-uncoupled state. The maintenance of oxidative stress seems to be important in melanoma apoptosis resistance because Mn(III)TBAP, a superoxide scavenger, or L-sepiapterin renders Tm5 cells more sensitive to anoikis and chemotherapy. More importantly, eNOS uncoupling seems to play a pivotal role in melanocyte malignant transformation induced by sustained anchorage impediment, because no malignant transformation was observed when L-NAME-treated melanocytes were subjected to sequential cycles of de-adhesion. Our results show that uncoupled eNOS contributes to superoxide production during melanocyte anchorage impediment, contributing to anoikis resistance and malignant transformation.