It is known that cyclooxygenase-2 (COX-2) inhibition elicits significant renal hemodynamics alterations when sodium intake is low. However, the mechanisms involved in these renal changes are not well known. Our objective was to evaluate the role of angiotensin II and 5-lipooxygenase-derived metabolites in the renal effects induced by prolonged COX-2 inhibition when sodium intake is low. Conscious dogs were treated during 7 days with a COX-2 inhibitor (1 mg·kg·d, SC75416), and either a vehicle, an AT1 receptor antagonist (0.4 mg · kg · d, candesartan) or a selective 5-lipooxygenase inhibitor (PF-150, 20 and 60 mg · kg · d). The administration of SC75416 alone induced significant changes in renal blood flow (219 ± 14 to 160 ± 10 mL/min), glomerular filtration rate (51 ± 2 to 42 ± 3 mL/min), and plasma potassium (pK) (4.3 ± 0.1 to 4.6 ± 0.1 mEq/L). Similar decrements in renal blood flow (27%) and glomerular filtration rate (20%) and a similar increment in pK (7%) were found when SC75416 was administered in candesartan-pretreated dogs. However, SC75416 administration did not elicit significant changes in renal hemodynamics and pK in dogs pretreated with each dose of PF-150. Our data suggest that leukotrienes but not angiotensin II are involved in the renal effects induced by COX-2 inhibition when sodium intake is low.
In recent study we found that treatment with an Ang II AT1 receptor antagonist (ARA) during nephrogenic period reduces nephron number in males and females but the subsequent renal structural changes are greater in males. The aim was to evaluate whether there are gender differences in the excretory response to an acute volume expansion (AVE) in rats with a decreased nephron number, and whether these differences are enhanced during aging. Newborn SD rats were treated with vehicle or an ARA (L-158.809, 7 mg/kg/day) during the first 14 postnatal days and the renal response to an AVE examined at three and nine months of age in control (CO) and ARA treated males and females. Arterial pressure and renal hemodynamics did not change in any group during the AVE. At three months of age, AVE-induced increments in sodium excretion and urine flow rate were greater in vehicle treated males (101 ± 4 μEq/min/bw and 620 ± 3 μl/min/bw) than in ARA treated males (54 ± 3 μEq/min/bw and 360 ± 4 μl/min/bw). Reduction in the renal response to AVE was similar in ARA treated males and females. Renal excretory ability to eliminate the AVE only was affected by age in ARA treated males. Natriuresis and diuresis decreased by 33% and 40%, respectively, in this group at nine vs. three months of age. The results of this study suggest that the renal excretory ability to eliminate an AVE is impaired when nephron number is reduced, and only aggravated by aging in males.
Luminal flow augments Na+ reabsorption in the thick ascending limb more than can be explained by increased ion delivery. This segment reabsorbs 30% of the filtered load of Na+, playing a key role in its homeostasis. Whether flow elevations enhance Na+-K+-2Cl- cotransporter (NKCC2) activity and the second messenger involved are unknown. We hypothesized that raising luminal flow augments NKCC2 activity by enhancing superoxide ([Formula: see text]) production by NADPH oxidase 4 (NOX4). NKCC2 activity was measured in thick ascending limbs perfused at either 5 or 20 nl/min with and without inhibitors of [Formula: see text] production. Raising luminal flow from 5 to 20 nl/min enhanced NKCC2 activity from 4.8 ± 0.9 to 6.3 ± 1.2 arbitrary fluorescent units (AFU)/s. Maintaining flow at 5 nl/min did not alter NKCC2 activity. The superoxide dismutase mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride blunted NKCC2 activity from 3.5 ± 0.4 to 2.5 ± 0.2 AFU/s when flow was 20 nl/min but not 5 nl/min. When flow was 20 nl/min, NKCC2 activity showed no change with time. The selective NOX1/4 inhibitor GKT-137831 blunted NKCC2 activity when thick ascending limbs were perfused at 20 nl/min from 7.2 ± 1.1 to 4.5 ± 0.8 AFU/s but not at 5 nl/min. The inhibitor also prevented luminal flow from elevating [Formula: see text] production. Allopurinol, a xanthine oxidase inhibitor, had no effect on NKCC2 activity when flow was 20 nl/min. Tetanus toxin prevents flow-induced stimulation of NKCC2 activity. We conclude that elevations in luminal flow enhance NaCl reabsorption in thick ascending limbs by stimulating NKCC2 via NOX4 activation and increased [Formula: see text]. NKCC2 activation is primarily the result of insertion of new transporters in the membrane.
Superoxide (O2−) exerts its physiological actions in part by causing changes in gene transcription. In thick ascending limbs flow-induced O2− production is mediated by NADPH oxidase 4 (Nox4) and is dependent on protein kinase C (PKC). Polymerase delta interacting protein 2 (Poldip2) increases Nox4 activity, but it is not known whether Nox4 translocates to the nucleus and whether Poldip2 participates in this process. We hypothesized that luminal flow causes Nox4 translocation to the nuclei of thick ascending limbs in a PKC-dependent process facilitated by Poldip2. To test our hypothesis, we studied the subcellular localization of Nox4 and Poldip2 using confocal microscopy and O2− production in the absence and presence of luminal flow. Luminal flow increased the ratio of nuclear to cytoplasmic intensity of Nox4 (N/C) from 0.3 ± 0.1 to 0.7 ± 0.1 (P < 0.01) and O2− production from 89 ± 15 to 231 ± 16 AU/s (P < 0.001). In the presence of flow PKC inhibition reduced N/C from 0.5 ± 0.1 to 0.2 ± 0.1 (P < 0.01). Flow-induced O2− production was also blocked (flow: 142 ± 20 AU/s; flow plus PKC inhibition 26 ± 12 AU/s; P < 0.01). The cytoskeleton disruptor cytochalasin D (1 μmol/L) decreased flow-induced Nox4 translocation by 0.3 ± 0.01 (P < 0.01); however, it did not reduce flow-induced O2−. Flow did not alter Poldip2 localization. We conclude that: (1) luminal flow elicits Nox4 translocation to the nucleus in a PKC- and cytoskeleton-dependent process; (2) Nox4 activation occurs before translocation; and (3) Poldip2 is not involved in Nox4 nuclear translocation. Flow-induced Nox4 translocation to the nucleus may play a role in O2−-dependent changes in thick ascending limbs.
Numerous studies have evaluated blood pressure (BP) and renal changes in several models of developmental programming of hypertension. The present study examined to what extent BP, renal hemodynamic, and renal structure are affected at an old age in male and female animals with altered renal development. It also evaluated whether renal damage is associated with changes in cyclooxygenase (COX)-2 and neuronal nitric oxide synthase (NOS1) expression and immunoreactivity. Experiments were carried out in rats at 10-11 and 16-17 mo of age treated with vehicle or an ANG II type 1 receptor antagonist during the nephrogenic period (ARAnp). A progressive increment in BP and a deterioration of renal hemodynamics were found in both sexes of ARAnp-treated rats, with these changes being greater (P < 0.05) in male rats. The decrease in glomerular filtration rate at the oldest age was greater (P < 0.05) in male (74%) than female (32%) ARAnp-treated rats. Sex-dependent deterioration of renal structure was demonstrated in optical and electron microscopic experiments. COX-2 and NOS1 immunoreactivity were enhanced in the macula densa of male but not female ARAnp-treated rats. The present study reports novel findings suggesting that stimuli that induce a decrease of ANG II effects during renal development lead to a progressive increment in BP and renal damage at an old age in both sexes, but these BP and renal changes are greater in males than in females. The renal damage is associated with an increase of COX-2 and NOS1 in the macula densa of males but not females with altered renal development.
Seminal plasma (SP) plays a crucial role in sperm protection and functionality. However, the effect of SP on the sperm cryopreservation is dependent on the stallion and SP composition. The use of epididymal spermatozoa incubated in the presence of SP could help the identification of the components of SP that are able to confer protection upon the spermatozoa during freezing.The aims of this study were (i) to identify SP components involved in the potential protection of epididymal spermatozoa during the freeze-thawing process and (ii) to identify and evaluate the proteins likely related to sperm freezability, using two-dimensional difference gel electrophoresis (2D-DIGE).Epididymal spermatozoa from 4 stallions were incubated with SP (80%, v/v) or without SP (control) before freezing. Sperm parameters were evaluated after thawing (viability, chromatin condensation, acrosomal integrity, reactive oxygen species [ROS]) and SP composition: total antioxidant capacity (TAC), fatty acid composition, total protein concentration, and protein components by 2D-DIGE.After thawing, the proportions of viable and acrosome-intact spermatozoa were higher than control when SP from two stallions was used (F and O). The SP of all stallions reduced ROS production in comparison with the control. After analyzing the SP components, it was found that total protein concentration, TAC, polyunsaturated fatty acids (PUFA), and eight specific proteins identified by 2D-DIGE were different between stallions.These studies allow the identification of SP components that could be involved in sperm protection or cryotolerance. Use of this information could help in the selection of stallions according to their semen freezing capacity.The composition of the SP probably contributes to semen cryotolerance capacity. Total protein, TAC, PUFA, and some proteins such as cysteine-rich secreted protein 3 could be used as biomarkers for the selection for sperm cryotolerance.
The G‐protein coupled receptor formyl peptide receptor 2 (FPR2) integrates signals of multiple anti‐inflammatory and anti‐fibrotic mediators including lipoxin A4, resolvin D1, and annexin A1. Previous studies have demonstrated protective effects of FPR2 in animal models of renal disease but the localization of FPR2 in the healthy kidney and its regulation in chronic kidney disease (CKD) have not been elucidated. Aim of this study was to determine the renal localization of FPR2 under control conditions and in a rat model of CKD. Newborn rats were treated with the AT1R antagonist Candesartan from postnatal d1‐14 to impair nephrogenesis and examined at 11 moth of age (CKD rats). Expression of FPR2 was studied using immunohistochemistry (IHC) and double labeling immunofluorescence (IF) for FPR2 and markers for fibroblasts, myofibroblasts, macrophages, and endothelial cells. CKD rats showed signs of renal inflammation and fibrosis with a focal accumulation of macrophages, myofibroblasts and extracellular matrix components. IHC revealed expression of FPR2 in the tubulointerstitium of controls and a strong accumulation of FPR2 expressing cells in fibrotic areas of CKD animals. IF identified fibroblasts and endothelial cells as the predominant FPR2 expressing cell types in both groups. Abundant signal was further detected in macrophages and myofibroblasts in the fibrotic areas of CKD animals. Our results show that FPR2 is abundantly expressed in the kidneys of control and CKD animals. It may therefore play an important role in the regulation of normal renal function and exert anti‐inflammatory and anti‐fibrotic effects during CKD. FPR2 may thus provide a promising target for pharmacological intervention in the treatment of CKD.
Abstract Amygdalin is a cyanogenic diglucoside and constitutes the bitter component in bitter almond (Prunus dulcis). Amygdalin concentration increases in the course of fruit formation. The monoglucoside prunasin is the precursor of amygdalin. Prunasin may be degraded to hydrogen cyanide, glucose, and benzaldehyde by the action of the β-glucosidase prunasin hydrolase (PH) and mandelonitirile lyase or be glucosylated to form amygdalin. The tissue and cellular localization of PHs was determined during fruit development in two sweet and two bitter almond cultivars using a specific antibody toward PHs. Confocal studies on sections of tegument, nucellus, endosperm, and embryo showed that the localization of the PH proteins is dependent on the stage of fruit development, shifting between apoplast and symplast in opposite patterns in sweet and bitter cultivars. Two different PH genes, Ph691 and Ph692, have been identified in a sweet and a bitter almond cultivar. Both cDNAs are 86% identical on the nucleotide level, and their encoded proteins are 79% identical to each other. In addition, Ph691 and Ph692 display 92% and 86% nucleotide identity to Ph1 from black cherry (Prunus serotina). Both proteins were predicted to contain an amino-terminal signal peptide, with the size of 26 amino acid residues for PH691 and 22 residues for PH692. The PH activity and the localization of the respective proteins in vivo differ between cultivars. This implies that there might be different concentrations of prunasin available in the seed for amygdalin synthesis and that these differences may determine whether the mature almond develops into bitter or sweet.
Medullary O 2 - production is elevated in Dahl salt-sensitive (SS) when compared to Dahl salt-resistant rats (SR), and this is one of the main contributors to salt-sensitive hypertension in this model. In thick ascending limbs (TALs), flow-induced O 2 - is caused by elevated ion delivery and cellular stretch. Mechanical stimulation by cellular stretch, in turn, leads to increases in intracellular calcium (Cai). We hypothesized that the elevated O 2 - production by SS TALs is due to greater stretch-induced increases in Cai mediated by Transient Receptor Potential Vanilloid (TRPV4). To test our hypothesis, we measured O 2 - and Cai in isolated, perfused TALs using the ratiometric dyes dihydroethidium and Fura2, respectively. Stretch led to a greater increase in Cai in SS (243±51 nM; n=9) compared to SR (124±27 nM; n=10; p<0.05 vs. SS). The increase in Cai and the difference between strains were blunted when tubules were treated with RN1734, a TRPV4 inhibitor (SS: 59±10 nM; SR: 24±3 nM; n=5 in each group). TRPV4 facilitates Ca influx into the cell. Thus we tested the effect of removing extracellular Ca on the response to stretch. When tubules were perfused and bathed with in Ca-free solutions stretch-induced increases in Cai and the difference between SS and SR TALs were completed eliminated (SS: 10±6 nM; SR: 8±4 nM; n=5 in each group). Transfecting SS TALs with an adenovirus expressing a TRPV4-small hairpin RNA abolished the difference in the stretch-induced Cai response between SS and SR tubules (SS: 75±15nM; SR: 56±28 nM; n=4 for each group). Stretch-induced O 2 - production was greater in SS TALs compared to SR tubules (SS: 59±10 AU/min; SR: 24±3 AU/min; p<0.02; n=5 for each group). The increase in O 2 - production caused by stretch and the difference between SS and SR TALs were eliminated when tubules were treated with the TRPV4 inhibitor RN1734 (SS: 15±7 AU/min; SR: 10±4 AU/min). Our results indicate that: 1) stretch increases Cai in SS and SR TALs and this is mediated by activation of TRPV4; 2) stretch raises Cai more in SS than SR tubules; 3) stretch-induced O 2 - production is elevated in SS TALs compared to those from SR and this is due to greater increases in Cai; and 4) differences in TRPV4 activation likely explain, in part, both the differences in stretch-induced Cai and O 2 - production.
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