Fumarate and its downstream signalling pathways in the cardiorenal system: Recent insights and novel expositions in the etiology of hypertension.

Hypertension, a risk factor for cardiorenal disease has a huge global health impact. Hence, there is a continuous search for new therapeutic targets and putative antihypertensive ligands. This search has transcended into the realm of mitochondrial metabolism which has been reported to underline the etiology of certain diseases, including hypertension. Recently, genetic alterations in the tricarboxylic acid (TCA) cycle enzyme, fumarase, which converts fumarate to malate, reportedly worsened salt-sensitive hypertension. These novel expositions shifted focus into the activity of TCA in the pathogenesis of hypertension. There is now evidence to show that a mechanistic link exists between blood pressure regulation and intermediaries in the TCA cycle involving fumarate metabolism. Fumarate has been reported to mediate the actions of endogenous ligands such as nitric oxide (NO), and hypoxia inducible factor (HIF)-1α. Similarly, there has been upregulation of protective genes such as nuclear erythroid factor 2 (Nrf2) and reduction in the expression of certain markers like kidney injury molecule 1 (KIM-1). There are reports of interactions with endogenous enzymes such as catalase (CAT) and renin via the activation of GPR91. Fumarate has also been shown to modulate the actions of renal ion channels and by extension, natriuresis. These actions of fumarate have conferred a reno- and cardio-protective effect in hypertension. This review evaluates the role of the TCA cycle, its mechanistic links, and significant contribution to blood pressure regulation with a view to understanding the possibility of a new pathological axis which may be involved in the pathogenesis of hypertension.

Malate reduced kidney injury molecule (KIM-1) expression and selectively upregulated the renal nitric oxide production in obstructive nephropathy.

BACKGROUND: Malate, the tricarboxylic acid (TCA) cycle intermediary, upregulates renal nitric oxide (NO) signaling, and NO is renoprotective in nephropathy. OBJECTIVES: This study explored the hypothesis that malate could increase renal NO and decrease renal injury and fibrotic markers in obstructive nephropathy. METHODS: Kidney injury was induced in rats via unilateral surgical ligation of the ureter, there after, rats were treated with malate (600 mg/kg, p.o.) for ten days. Urine was collected on days 0, 4, 7 and 10. Urinary sodium excretion was also determined. Western blot and biochemical analyses were carried on the nephropathic kidneys. RESULTS: Malate reduced kidney injury molecule (KIM-1) expression in the renal cortex and medulla of nephropathic rats (p < 0.05). NO production was selectively increased in the medulla of nephropathic rats treated with malate (58.3 ± 1.3 vs 77.8 ± 4.4 µM/ng, p < 0.05). Superoxide dismutase and catalase activity increased in the kidney of malate-treated nephropathic rats (p < 0.05). Transforming growth factor (TGF-ß), an index of fibrosis, increased in the cortex but not medulla of the malate-treated UUO group. There was a consistent increase in collagenase activity in the cortex, and a reduction in the medulla. CONCLUSION: Malate ameliorated the injury and inflammation but selectively reduced fibrosis in obstructive nephropathy (Fig. 6, Ref. 32). Text in PDF www.elis.sk Keywords: Malate, tricarboxylic acid cycle, nitric oxide, kidney injury molecule (KIM-1), obstructive nephropathy.

Malate reduced blood pressure and exerted differential effects on renal hemodynamics; role of the nitric oxide system and renal epithelial sodium channels (ENaC).

Malate regulates blood pressure via nitric oxide production in salt-sensitive rats, a genetic model of hypertension. This study investigated the possible contributions of malate to blood pressure regulation and renal haemodynamics in normotensive rats. Malate (0.1, 0.3 and 1 µg/kg, iv) was injected into rats or L-nitro-arginine methyl ester (L-NAME)-treated rats and mean arterial blood pressure (MABP), cortical blood flow (CBF), and medullary blood flow (MBF), was measured. The clearance study involved infusion of malate at 0.1 µg/kg/h into rats, and MABP, CBF, MBF, glomerular filtration rate (GFR), urine volume (UV) and sodium output (UNaV) were determined. Mechanistic studies to evaluate the role of renal sodium channels involved the treatment with malate (600 mg/kg, po), amiloride (2.5 mg/kg, po) or hydrochlorothiazide (HCTZ) (10 mg/kg, po), and UV and UNaV were determined. Malate elicited significant peak reductions in MABP (124 ± 6.5 vs 105 ± 3.1 mmHg) at 0.1 µg/kg), CBF (231 ± 18.5 vs 205 ± 10.9 PU). L-NAME did not reverse the effect of malate on MABP but tended to blunt the effect on CBF (40%) and MBF (87%) at 0.3 µg/kg. Infusion of malate reduced MABP, CBF, and MBF in a time-dependent manner (p<0.05). Malate exerted a three-fold decrease in GFR in a time-related fashion (p<0.05) as well as increased UV. UNaV increased by 86% in malate-treated-amiloride rats (p<0.05). These data indicate that malate modulates blood pressure and exerts vascular and tubular effects on renal function that may involve epithelial sodium channels (ENaC).

Fumarate exerted an antihypertensive effect and reduced kidney injury molecule (KIM)-1 expression in deoxycorticosterone acetate-salt hypertension.

Background: The tricarboxylic (TCA) acid cycle provides the energy needed for regulatory functions in the cardio-renal system. Recently, a genetic defect in the TCA cycle enzyme, fumarase hydratase, altered L-arginine metabolism and exacerbated hypertension in salt-sensitive rats. This study evaluated the effect of fumarate and its possible link to L-arginine metabolism in deoxycorticosterone (DOCA)-salt hypertension, a non-genetic model of hypertension.Method: Hypertension was induced with DOCA (25 mg/kg s.c, twice weekly) + 1% NaCL in uninephrectomised rats placed on fumarate (1 g/L, ad libitum). Blood pressure was measured in conscious rats via carotid cannulation. Biochemical and western blot analyses were carried out on kidney fractions.Results: Fumarate reduced mean blood pressure (198 ± 5 vs 167 ± 7 mmHg, p < .01), increased nitric oxide levels in the renal cortex (36.1 ± 2 vs 61.3 ± 4 nM/µg) and medulla (27.4 ± 1 vs 54.1 ± 2 nM/µg) of DOCA-salt rats (p < .01). Consistent with this, arginase activity was reduced (threefold) in the renal medulla but not cortex of DOCA-salt rats. Fumarate increased superoxide dismutase activity in the medulla (p < .001) of DOCA-hypertensive rats. However, catalase activity was exacerbated by fumarate in both renal cortex (4.5 ± 1 vs 11.2 ± 1) and medulla (3.7 ± 1 vs 16.3 ± 1 units/mg) of DOCA-salt rats (p < .001). Proteinuria (64.6%), kidney injury molecule-1 expression and kidney weight were reduced in DOCA-hypertensive rats treated with fumarate (p< .05). However, there was a paradoxical increase in TGF-ß expression in fumarate-treated DOCA-salt rats. Conclusion: These data show that fumarate attenuated hypertension, renal injury and improved the redox state of the kidney in DOCA/salt hypertension by mechanisms involving selective reduction of L-arginine metabolism.

The Research Centers in Minority Institutions (RCMI) Translational Research Network: Building and Sustaining Capacity for Multi-Site Basic Biomedical, Clinical and Behavioral Research.

The Research Centers in Minority Institutions (RCMI) program was established by the US Congress to support the development of biomedical research infrastructure at minority-serving institutions granting doctoral degrees in the health professions or in a health-related science. RCMI institutions also conduct research on diseases that disproportionately affect racial and ethnic minorities (ie, African Americans/Blacks, American Indians and Alaska Natives, Hispanics, Native Hawaiians and Other Pacific Islanders), those of low socioeconomic status, and rural persons. Quantitative metrics, including the numbers of doctoral science degrees granted to underrepresented students, NIH peer-reviewed research funding, peer-reviewed publications, and numbers of racial and ethnic minorities participating in sponsored research, demonstrate that RCMI grantee institutions have made substantial progress toward the intent of the Congressional legislation, as well as the NIH/NIMHD-linked goals of addressing workforce diversity and health disparities. Despite this progress, nationally, many challenges remain, including persistent disparities in research and career development awards to minority investigators. The continuing underrepresentation of minority investigators in NIH-sponsored research across multiple disease areas is of concern, in the face of unrelenting national health inequities. With the collaborative network support by the RCMI Translational Research Network (RTRN), the RCMI community is uniquely positioned to address these challenges through its community engagement and strategic partnerships with non-RCMI institutions. Funding agencies can play an important role by incentivizing such collaborations, and incorporating metrics for research funding that address underrepresented populations, workforce diversity and health equity.

Regulation of hypoxia inducible factor/prolyl hydroxylase binding domain proteins 1 by PPARα and high salt diet.

BACKGROUND: Hypoxia inducible factor (HIF)/prolyl hydroxylase domain (PHD)-containing proteins are involved in renal adaptive response to high salt (HS). Peroxisome proliferator activated receptor alpha (PPARα), a transcription factor involved in fatty acid oxidation is implicated in the regulation of renal function. As both HIF-1α/PHD and PPARα contribute to the adaptive changes to altered oxygen tension, this study tested the hypothesis that PHD-induced renal adaptive response to HS is PPARα-dependent. METHODS: PPARα wild type (WT) and knock out (KO) mice were fed a low salt (LS) (0.03% NaCl) or a HS (8% NaCl) diet for 8 days and treated with hydralazine. PPARα and heme oxygenase (HO)-1 expression were evaluated in the kidney cortex and medulla. A 24-h urinary volume (UV), sodium excretion (UNaV), and nitrite excretion (UNOx V) were also determined. RESULTS: PHD1 expression was greater in the medulla as compared to the cortex of PPARα WT mice (p<0.05) fed with a LS (0.03% NaCl) diet. The HS diet (8% NaCl) downregulated PHD1 expression in the medulla (p<0.05) but not the cortex of WT mice whereas expression was downregulated in the cortex (p<0.05) and medulla (p<0.05) of KO mice. These changes were accompanied by HS-induced diuresis (p<0.05) and natriuresis (p<0.05) that were greater in WT mice (p<0.05). Similarly, UNOx V, index of renal nitric oxide synthase (NOS) activity or availability and heme oxygenase (HO)-1 expression was greater in WT (p<0.05) but unchanged in KO mice on HS diet. Hydralazine, a PHD inhibitor, did not affect diuresis or natriuresis in LS diet-fed WT or KO mice but both were increased (p<0.05) in HS diet-fed WT mice. Hydralazine also increased UNOx V (p<0.05) with no change in diuresis, natriuresis, or HO-1 expression in KO mice on HS diet. CONCLUSIONS: These data suggest that HS-induced PPARα-mediated downregulation of PHD1 is a novel pathway for PHD/HIF-1α transcriptional regulation for adaptive responses to promote renal function via downstream signaling involving NOS and HO.

Role of mechanistic target of rapamycin (mTOR) in renal function and ischaemia-reperfusion induced kidney injury.

Despite the presence of many studies on the role of mechanistic target of rapamycin (mTOR) in cardiorenal tissues, the definitive role of mTOR in the pathogenesis of renal injury subsequent to ischaemia-reperfusion (IR) remains unclear. The aims of the current study were to characterize the role of mTOR in normal kidney function and to investigate the role of mTOR activation in IR-induced kidney injury. In euvolemic anaesthetized rats, treatment with the mTOR inhibitor rapamycin increased blood pressure (121 ± 2 to 144 ± 3 mmHg; P<.05), decreased glomerular filtration rate (GFR; 1.6 ± 0.3 to 0.5 ± 0.2 mL/min; P<.05) and increased urinary sodium excretion (UNaV; 14 ± 1 to 109 ± 25 mmol/L per hour; P<.05). In rats subjected to IR, autophagy induction, p-mTOR expression and serum creatinine increased (1.9 ± 0.2 to 3 ± 0.3 mg/dL; P<.05); treatment with rapamycin blunted p-mTOR expression but further increased autophagy induction and serum creatinine (3 ± 0.3 to 5 ± 0.6 mg/dL; P<.05). In contrast, clenbuterol, an mTOR activator, blunted the effect of rapamycin on serum creatinine (4 ± 0.6 vs 2.3 ± 0.3 mg/dL; P<.05), autophagy induction and p-mTOR expression. IR also increased 24 hour protein excretion (9 ± 3 to 17 ± 2 mg/day; P<.05) and kidney injury molecule-1 (KIM-1) expression, and rapamycin treatment further increased KIM-1 expression. Clenbuterol exacerbated protein excretion (13 ± 2 to 26 ± 4 mg/day; P<.05) and antagonized the effect of rapamycin on KIM-1 expression. Histopathological data demonstrated kidney injury in IR rats that was worsened by rapamycin treatment but attenuated by clenbuterol treatment. Thus, mTOR signalling is crucial for normal kidney function and protecting the kidney against IR injury through autophagy suppression.

The Role of Hypoxia-Inducible Factor/Prolyl Hydroxylation Pathway in Deoxycorticosterone Acetate/Salt Hypertension in the Rat.

KKidney disease could result from hypertension and ischemia/hypoxia. Key mediators of cellular adaptation to hypoxia are oxygen-sensitive hypoxia inducible factor (HIF)s which are regulated by prolyl-4-hydroxylase domain (PHD)-containing dioxygenases. However, HIF activation can be protective as in ischemic death or promote renal fibrosis in chronic conditions. This study tested the hypothesis that increased HIF-1α consequent to reduced PHD expression contributes to the attendant hypertension and target organ damage in deoxycorticosterone acetate (DOCA)/salt hypertension and that PHD inhibition ameliorates this effect. In rats made hypertensive by DOCA/salt treatment (DOCA 50 mg/kg s/c; 1% NaCl orally), PHD inhibition with dimethyl oxallyl glycine (DMOG) markedly attenuated hypertension (P<0.05), proteinuria (P<0.05) and attendant tubular interstitial changes and glomerular damage (P<0.05). Accompanying these changes, DMOG blunted the increased expression of kidney injury molecule (KIM)-1 (P<0.05), a marker of tubular injury and reversed the decreased expression of nephrin (P<0.05), a marker of glomerular injury. DMOG also decreased collagen I staining (P<0.05), increased serum nitrite (P<0.05) and decreased serum 8-isopostane (P<0.05). However, the increased HIF-1α expression (P<0.01) and decreased PHD2 expression (P<0.05) in DOCA/salt hypertensive rats was not affected by DMOG. These data suggest that reduced PHD2 expression with consequent increase in HIF-1α expression probably results from hypoxia induced by DOCA/salt treatment with the continued hypoxia and reduced PHD2 expression evoking hypertensive renal injury and collagen deposition at later stages. Moreover, a PHD inhibitor exerted a protective effect in DOCA/salt hypertension by mechanisms involving increased nitric oxide production and reduced production of reactive oxygen species.

Interactions of PPAR-alpha and adenosine receptors in hypoxia-induced angiogenesis.

Hypoxia and adenosine are known to upregulate angiogenesis; however, the role of peroxisome proliferator-activated receptor alpha (PPARα) in angiogenesis is controversial. Using transgenic Tg(fli-1:EGFP) zebrafish embryos, interactions of PPARα and adenosine receptors in angiogenesis were evaluated under hypoxic conditions. Epifluorescent microscopy was used to assess angiogenesis by counting the number of intersegmental (ISV) and dorsal longitudinal anastomotic vessel (DLAV) at 28 h post-fertilization (hpf). Hypoxia (6h) stimulated angiogenesis as the number of ISV and DLAV increased by 18-fold (p<0.01) and 100 ± 8% (p<0.001), respectively, at 28 hpf. Under normoxic and hypoxic conditions, WY-14643 (10 µM), a PPARα activator, stimulated angiogenesis at 28 hpf, while MK-886 (0.5 µM), an antagonist of PPARα, attenuated these effects. Compared to normoxic condition, adenosine receptor activation with NECA (10 µM) promoted angiogenesis more effectively under hypoxic conditions. Involvement of A2B receptor was implied in hypoxia-induced angiogenesis as MRS-1706 (10nM), a selective A2B antagonist attenuated NECA (10 µM)-induced angiogenesis. NECA- or WY-14643-induced angiogenesis was also inhibited by miconazole (0.1 µM), an inhibitor of epoxygenase dependent production of eicosatrienoic acid (EET) epoxide. Thus, we conclude that: activation of PPARα promoted angiogenesis just as activation of A2B receptors through an epoxide dependent mechanism.

Inhibition of prolyl hydroxylase domain-containing protein on hypertension/renal injury induced by high salt diet and nitric oxide withdrawal.

BACKGROUND: Nitric oxide just as prolyl hydroxylase domain-containing protein (PHD) is a regulator of hypoxia inducible factor-1 α (HIF-1α), a transcription factor complex that controls the expression of most genes involved in hypoxia and cardiovascular diseases. In the absence of nitric oxide, it is not clear how HIF-1α and PHD are regulated and to what extent they contribute to the ensuing disorder. METHOD: Using the nitric oxide withdrawal/high salt diet model of hypertensive renal injury, this study tested the hypothesis that removal of the inhibition by nitric oxide on PHD predisposes to increased PHD but reduced HIF-1α expression, hypertension and renal injury. RESULTS: In animals treated with N-nitro-L-arginine (L-NNA; 250 mg/l in drinking water for 14 days) and high salt diet (4% NaCl), there was hypertension (41±5%, P<0.05), proteinuria (three-fold, P<0.05), kidney (22±3%, P<0.05) and heart enlargement (24±3%, P<0.05), as well as increased renal osteopontin (21±3%, P<0.05) and collagen IV (24±4%, P<0.05) expression. Accompanying these effects were increased expression of PHD1 (24±4%, P<0.05) and PHD2 (36±4%, P<0.05) but reduced HIF-1α (35±6%, P < 0.05) expression. Dimethyloxallyl glycine (5mg/kg), a PHD inhibitor, paradoxically exacerbated hypertension (46±7%, P<0.05), proteinuria (two-fold, P <0.05), and increased osteopontin (15±2%, P<0.05) and HIF-1α (31±5%, P<0.05) expression with no change in PHD1/2 expression or kidney and heart enlargement. CONCLUSION: These data suggest that the protective effect of physiological levels of nitric oxide may be by virtue of inhibition of PHD or increased HIF-1α expression, hence, the pathological changes produced following its withdrawal was accompanied by increased PHD or decreased HIF-1α expression. Exacerbation of hypertension and renal injury following PHD inhibition suggests a deleterious effect in the chronic setting and challenges the dogma that inhibition of PHD is useful in cardiovascular diseases.

Role of GLUT4 on angiotensin 2-induced systemic and renal hemodynamics.

Cross-talk between insulin and the renin angiotensin system signaling system shows that angiotensin 2 (A2) negatively modulates insulin signaling by stimulating multiple serine phosphorylation events in the early stages of the insulin-signaling cascade; however, the biological actions of A2 on insulin sensitivity remain controversial. Preservation of glucose transporter 4 (GLUT4) expression during hypertension has been shown to prevent the increased vascular reactivity associated with hypertension. This study tested the hypothesis that GLUT4 contributes to the renal actions of A2. In the euvolemic anesthetized rat, acute infusion of the GLUT4 antagonist, indinavir (1 mg/kg/minute), enhanced an A2-induced increase in mean arterial blood pressure (MABP) (P < 0.01), but attenuated an A2-induced increase in medullary blood flow (MBF) and glomerular filtration rate (P < 0.01). Insulin, a GLUT4 activator (20 mU/kg/minute and 40 mU/kg/minute), decreased basal MABP and urine volume (P < 0.05), but it increased MBF, and these effects were reversed and blunted by indinavir. Subchronic indinavir treatment (80 mg/kg/day orally for 15 days) did not affect A2-induced changes in MABP, cortical blood flow, and MBF, but significantly decreased basal MBF (P < 0.01) and global kidney perfusion (P < 0.05). We concluded that acute but not subchronic inhibition of GLUT4 alters A2-induced changes in systemic and renal hemodynamics by attenuating A2-induced increase in MBF and glomerular filtration rate.

PPARs and their effects on the cardiovascular system.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily that undergo transactivation or transrepression by distinct mechanisms leading to induction or repression of expression of target genes. The PPAR family consists of three isoforms, α, γ, and ß/δ, which share similar structural organization, possess distinct functions, and vary in their ligand affinity, expression, and activity in different metabolic pathways in different tissues. PPARs are involved in many functions especially those involved in the regulation of vascular tone, inflammation and energy homeostasis and therefore represent important targets for hypertension, obesity, obesity-induced inflammation, and metabolic syndrome in general. PPARs may influence the inflammatory response either by direct transcriptional downregulation of proinflammatory genes via mechanisms involving transrepression, or indirectly via their transcriptional effects on lipid metabolism. On account of their pleiotropic effects, they are now known to be active participants in many disease conditions and they represent potent targets for the development of therapy of a wide array of diseases.

Renal function and vasomotor activity in mice lacking the Cyp4a14 gene.

The production of 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney is thought to be involved in the control of renal vascular tone and tubular sodium and chloride reabsorption. Cytochrome (Cyp) P-450 enzymes of the Cyp4a family in the mouse, namely 4a10, -12 and 14, are involved in 20-HETE synthesis. Recent advances in the molecular genetics of the mouse have produced mice in which Cyp4a isoforms have been disrupted and the consequence of such an approach is examined. This study evaluated the effect of deletion of the Cyp4a14 gene on blood pressure, renal vascular responses and tubular function. When compared with the wild-type (WT) litter mates, systolic blood pressure was greater in Cyp4a14 null (KO) mice as were renal vascular responses to angiotensin II or phenyephrine, G protein-coupled receptor (GPCR) agonists, but not KCl, a non-GPCR agonist. Renal vascular responses to guanosine 5'-O-(gamma-thio)triphosphate, a non-hydrolyzable GTP analog, or NaF(4), an activator of G-proteins, were also enhanced. However, vasodilation to bradykinin or apocynin but not sodium nitroprusside was blunted in Cyp4a14 null (KO) kidneys. These changes in KO mice were accompanied by increased 20-HETE synthesis, reduced renal production of nitric oxide (NO), increased lipid hydroperoxides and increased apocynin-inhibitable vascular NADPH oxidase activity that was prevented by administration of NO synthase (NOS) inhibitor, suggesting endothelial nitric oxide synthase (eNOS) uncoupling. Cyp4a14 KO mice also exhibited a diminished capacity to excrete an acute sodium load (0.9% NaCl, 2.5 mL/kg). These data suggest that deletion of the Cyp4a gene conferred a prohypertensive status via mechanisms involving increased 20-HETE synthesis and eNOS uncoupling leading to increased oxidative stress, enhanced vasoconstriction but diminished vasodilation as well as a defect in the renal excretory capacity in Cyp4a14 KO mice. These mechanisms suggest that the Cyp4a14-deficient mouse may be a useful model for evaluation of NO/20-HETE interactions.

Regulation of cerebrovascular endothelial peroxisome proliferator activator receptor alpha expression and nitric oxide production by clofibrate.

BACKGROUND: Peroxisome proliferator activator receptor alpha (PPAR alpha), a member of the nuclear receptor superfamily, is known to increase nitric oxide (NO) production and the mechanisms by which PPAR alpha activation alleviates vascular dysfunction may predicate its activation and possible expression. OBJECTIVES: We have evaluated the effects of acute clofibrate, a PPAR alpha ligand and the role of PKC on PPAR alpha expression and NO production in cultured cerebral microvascular endothelial cell (CMVEC). METHODS: Confluent CMVEC derived from pig brain were cultured and the role of PKC in acute clofibrate-induced PPAR alpha expression and NO production was determined in the presence or absence of PKC activator phorbol myristate acetate (PMA) or inhibitor (calphostin C). RESULTS: Incubation of CMVEC with clofibrate or PMA increased NO production by 40% or 27%, respectively, whereas co-incubation of cells with PMA and clofibrate had no effect on NO production. Incubation of cells with Calphostin C blunted PMA but not clofibrate-induced increase in NO production. L-NAME (0.1 mM), an inhibitor of NO synthase, reduced basal (47%; p<0.01) and abolished clofibrate-induced increase in NO production. Clofibrate increased PPAR alpha expression (26%; p<0.05) while PMA with or without clofibrate reduced PPAR alpha expression (p<0.01). On the other hand, calphostin C reduced basal (69%, ap<0.01) as well as clofibrate-induced increase (59%, p<0.01) in PPAR expression, and further reduced PMA-induced down regulation of PPAR expression. eNOS expression was not significantly affected by either clofibrate or PMA, alone or in combination. CONCLUSION: These results show that in the brain microvascular endothelial cell, PPAR alpha activation increases NO production-independent of eNOS and PKC signaling pathways, a regulates PPAR alpha expression through a complex PKC signaling mechanism(s) as both PKC activation and inhibition reduced clofibrate-induced activation of PPAR expression (Fig. 4, Ref. 32). Full Text (Free, PDF) www.bmj.sk.

Natriuretic and renoprotective effect of chronic oral neutral endopeptidase inhibition in acute renal failure.

Neutral endopeptidase (NEP: EC 3.4.24.11) is involved in the degradation of peptides such as atrial natriuretic peptide, angiotensin II (AngII), and endothelin-1 (ET-1). In this study we propose that NEP inhibition provides protection in glycerol-induced acute renal failure (ARF). Renal vascular responses were evaluated in ARF rats where ARF was induced by injecting 50% glycerol in candoxatril, a NEP inhibitor (30 mg/kg, orally; for 3 weeks) pretreated rats. AngII and U46619 (a TxA2 mimetic) vasoconstriction was increased (2- to 4-fold) in ARF while ET-1 vasoconstriction was surprisingly reduced (23+/-3%; p<0.05). In ARF, candoxatril paradoxically enhanced ET-1 response (60+/-20%; p<0.05) but reduced AngII vasoconstriction (51+/-11%; p<0.05) without affecting U46619 response. However, candoxatril treatment was without effect on plasma ET-1 and TxB2 levels in ARF. Candoxatril reduced plasma AngII by 34+/-4% (p<0.05) in ARF which was approximately 3.5-fold higher compared to control. Candoxatril doubled the nitrite excretion in control but was without effect on proteinuria or nitrite excretion in ARF. Candoxatril enhanced Na+ and creatinine excretion in ARF by 73+/-9% and 33+/-2%, respectively. These results suggest that NEP inhibition may confer protection in glycerol-induced ARF by stimulating renal function but without a consistent effect on renal production and renal vascular responses to endogenous vasoconstrictors.

Interaction of oxidative stress, nitric oxide and peroxisome proliferator activated receptor gamma in acute renal failure.

Oxidative stress has been reported to play a critical role in the pathology of acute renal failure (ARF). An interaction between different reactive species and/or their sources have been the focus of extensive studies. The exact sources of reactive species generated in biological systems under different disease states are always elusive because they are also a part of physiological processes. Exaggerated involvement of different oxidation pathways including NAD(P)H oxidase has been proposed in different models of ARF. An interaction between oxygen species and nitrogen species has drawn extensive attention because of the deleterious effects of peroxynitrite and their possible effects on antioxidant systems. Recent advances in molecular biology have allowed us to understand glomerular function more precisely, especially the organization and importance of the slit diaphragm. Identification of slit diaphragm proteins came as a breakthrough and a possibility of therapeutic manipulation in ARF is encouraging. Transcriptional regulation of the expression of slit diaphragm protein is of particular importance because their presence is crucial in the maintenance of glomerular function. This review highlights the involvement of oxidative stress in ARF, sources of these reactive species, a possible interaction between different reactive species, and involvement of PPARgamma, a nuclear transcription factor in this process.

Effect of peroxisome proliferator-activated receptor-alpha siRNA on hypertension and renal injury in the rat following nitric oxide withdrawal and high salt diet.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-alpha has been implicated in the regulation of normal and pathological cellular functions, but the effect of specific gene silencing on PPARalpha-mediated function is not fully defined. AIM: This study evaluated the role of PPARalpha in hypertensive renal injury induced by nitric oxide withdrawal and high salt (4% NaCl) diet [high salt/N(omega)-nitro-L-arginine (L-NNA)]. METHODS: Three PPARalpha siRNA clones, siRNA(790-811), siRNA(974-995) or siRNA(1410-1431), directed at the DNA or ligand binding domain of PPARalpha mRNA or scrambled siRNA was cloned into plasmid expression vector and was injected (10 microg intravenously) in hypertensive rats. Twenty-four-hour readings of blood pressure and heart rate were taken in conscious rats using radiotelemetry. Kidney injury was evaluated by determining N-acetyl-beta-glucosaminidase excretion, expression of kidney injury molecule-1 and histopathology. PPARalpha mRNA and protein expression were also determined. RESULTS: High salt/L-NNA increased PPARalpha mRNA expression three-fold, and this was abolished in rats treated with PPARalpha siRNA(790-811), siRNA(974-995) or siRNA(1410-1431). High salt/L-NNA also increased blood pressure but reduced heart rate without affecting pulse pressure. However, blood pressure was further increased in rats treated with PPARalpha siRNA(790-811) (37 +/- 3%, P < 0.05). High salt/L-NNA also increased N-acetyl-beta-glucosaminidase excretion and expression of kidney injury molecule-1. However, PPARalpha siRNA(790-811) did not affect N-acetyl-beta-glucosaminidase excretion but reduced kidney injury molecule-1 expression. Histopathology of kidney tissues in high salt/L-NNA-treated rats revealed global, fibrinoid and tubular interstitial necrosis that was blunted by PPARalpha siRNA(790-811). CONCLUSION: These data suggest that increased PPARalpha expression is a protective mechanism in hypertensive renal injury induced by nitric oxide withdrawal/high salt diet and that siRNAs targeting the DNA-binding domain of PPARalpha gene elicited differential effects on hypertension and kidney injury.

Role of G protein-coupled receptor kinase-2 in peroxisome proliferator-activated receptor gamma-mediated modulation of blood pressure and renal vascular reactivity in SHR.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear transcription factor, modulates the expression/activity of G protein-coupled receptors (GPCRs), but its role in GPCR signaling is not clear. Increased GPCR kinase-2 (GRK-2) activity and receptor desensitization have been reported in hypertension. METHOD: In this study we investigated the role of GRK-2 in PPARgamma-mediated blood pressure regulation in hypertension. SHR or WKY rats were treated with GW1929, a selective PPARgamma ligand (0.5 mg/kg/day), or vehicle for 2 months. Systolic blood pressure (tail cuff plethysmography), whole kidney perfusion (laser scanner) and renal vascular reactivity (isolated perfused kidney) was determined. RESULTS: GW1929 significantly reduced blood pressure (20 +/- 1%) and increased renal perfusion (61 +/- 3%) in SHR compared to WKY rats. Vasoconstriction to phenylephrine (100 microg) in the isolated perfused kidney was greater in SHRs (29 +/- 1%) compared to WKY rats and this was abolished by GW1929. GW1929 enhanced acetylcholine-induced (30-300 microg) and sodium nitroprusside-induced vasodilatation in SHR by 46 +/- 2% (p < 0.05) and 33 +/- 2% (p < 0.05), respectively. Isoprenalin-induced (5-30 microg) vasodilatation was 43 +/- 2% lower in SHR compared to WKY and GW1929 enhanced this vasodilatation by 55 +/- 2%. In SHR kidney, GW1929 enhanced expression of PPARgamma mRNA (34 +/- 1%) but reduced that of GRK-2 (31 +/- 3%). CONCLUSION: We suggest that downregulation of PPARgamma but upregulation of GRK-2 increases blood pressure and impaired renal vascular reactivity in SHR and that PPARgamma-mediated improvement in hypertension may involve transcriptional regulation of GRK-2 function.

Regulation of blood pressure, natriuresis and renal thiazide/amiloride sensitivity in PPARalpha null mice.

This study evaluated the role of PPARalpha in renal function and whether PPARalpha knockout (KO) mice are hypertensive or salt-sensitive. We hypothesize that PPARalpha modulation of ion transport defines the capacity for sodium excretion (U(Na)V). PPARalpha KO and wild-type (WT) mice were placed on a normal salt (NS, 0.5% NaCl) or high salt (8% NaCl, HS) diet for 28 days and mean arterial blood pressure (MABP) and heart rate (HR) determined. In a group of anesthetized animals on NS diet, pressure natriuresis (P/N) was determined and in another group, acute sodium load (0.9% NaCl) was administered and U(Na)V compared in mice pretreated with amiloride (200 microg/kg) or hydrochlorothiazide (3 mg/kg), in vivo measurements of sodium hydrogen exchanger or Na-Cl-cotransporter activity, respectively. MABP and HR were similar in PPARalpha KO and WT mice placed on a NS diet (116+/-6 mmHg, 587+/-40 beats/min, KO; 116+/-4 mmHg, 551+/-20 beats/min, WT). HS diet increased MABP to a greater extent in KO mice (Delta = 29+/-3 vs 14+/-3 mmHg, p<0.05) as did proteinuria (8- vs 2.5-fold, p<0.05). P/N was blunted in untreated KO mice. In response to an acute NaCl-load, U(Na)V was faster in PPARalpha KO mice (4.31+/-1.11 vs 0.77+/-0.31 micromol, p<0.05). However, U(Na)V was unchanged in hydrochlorothiazide-treated KO mice but increased 6.9-fold in WT mice. Similarly, U(Na)V was less in amiloride-treated KO mice (3.4- vs 15.5-fold). These data suggest that PPARalpha participates in pressure natriuresis and affects Na transport via amiloride- and thiazide-sensitive mechanisms. Thus, despite defective fatty acid oxidation, PPARalpha null mice are not hypertensive but develop salt-sensitive hypertension.

Peroxisome proliferator-activated receptor alpha activation attenuated angiotensin type 1-mediated but enhanced angiotensin type 2-mediated hemodynamic effects to angiotensin II in the rat.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent nuclear transcription factors that regulate beta-oxidation of fatty acids in various tissues. PPARalpha ligands also protect against pathological damage especially resulting from angiotensin II hypertension. The modulating effect of PPARalpha on hemodynamic effects elicited by angiotensin II under normal conditions, however, is not fully known. METHOD: We therefore evaluated renal and systemic hemodynamic effects of angiotensin II in normal animals treated with PPARalpha ligands. RESULTS: PPARalpha ligands clofibrate (250 mg/kg), fenofibrate (100 mg/kg), or pirixinic acid (WY14643; 45 mg/kg) each elicited an increase in renal peroxisomal beta-oxidation, accompanied by increased renal nitric oxide production. Clofibrate blunted the angiotensin II (3-100 ng/kg)-induced increase in mean arterial blood pressure (P < 0.05) but attenuated the reduction in renal cortical blood flow (laser Doppler flowmetry; P < 0.05). N(omega)-nitro-L-arginine methyl ester (L-NAME) but not D-NAME (100 mg/l) blunted clofibrate-induced inhibition of angiotensin II responses. In the presence of the angiotensin type 1 (AT1)-antagonist losartan (3 mg/kg), clofibrate uncovered a hypotensive effect of angiotensin II and further blunted the residual renal vasoconstriction. L-NAME or the angiotensin type 2 (AT2)-antagonist (S-[+]-1-[(4-dimethylamino]-3-methylphenyl)methyl]-5-[diphenylacetyl]-4,5,6,7-tetrahydro-1H-imidazol[4,5-c]pyridine-6-carboxilic acid; PD123319), but not D-NAME, blunted the effects of losartan and blocked the hypotensive effects of angiotensin II in losartan-treated rats. Except in rats treated for 7 days with WY14643, AT1-receptor expression was downregulated (P < 0.05) while AT2-receptor expression was upregulated (P < 0.05) in renal cortical homogenates from rats treated with clofibrate or WY14643. CONCLUSION: These data suggest that PPARalpha activation counters AT1-mediated pressor and vasoconstrictor effects and that, during AT1 receptor blockade, PPARalpha activation leads to hypotension coupled to AT2-receptor activation by a mechanism probably involving nitric oxide production.