Super-resolved local recruitment of CLDN5 to filtration slits implicates a direct relationship with podocyte foot process effacement.

Under healthy conditions, foot processes of neighbouring podocytes are interdigitating and connected by an electron-dense slit diaphragm. Besides slit diaphragm proteins, typical adherens junction proteins are also found to be expressed at this cell-cell junction. It is therefore considered as a highly specialized type of adherens junction. During podocyte injury, podocyte foot processes lose their characteristic 3D structure and the filtration slits typical meandering structure gets linearized. It is still under debate how this change of structure leads to the phenomenon of proteinuria. Using super-resolution 3D-structured illumination microscopy, we observed a spatially restricted up-regulation of the tight junction protein claudin-5 (CLDN5) in areas where podocyte processes of patients suffering from minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) as well as in murine nephrotoxic serum (NTS) nephritis and uninephrectomy DOCA-salt hypertension models, were locally injured. CLDN5/nephrin ratios in human glomerulopathies and NTS-treated mice were significantly higher compared to controls. In patients, the CLDN5/nephrin ratio is significantly correlated with the filtration slit density as a foot process effacement marker, confirming a direct association of local CLDN5 up-regulation in injured foot processes. Moreover, CLDN5 up-regulation was observed in some areas of high filtration slit density, suggesting that CLND5 up-regulation preceded the changes of foot processes. Therefore, CLDN5 could serve as a biomarker predicting early foot process effacement.

Epac-mediated relaxation in murine basilar arteries depends on membrane permeability of cyclic nucleotide analogues and endothelial aging.

Cerebral blood supply is finely tuned by regulatory mechanisms depending on vessel caliber the disruption of which contributes to the development of diseases such as vascular dementia, Alzheimer's and Parkinson 's diseases. This study scopes whether cAMP-mimetic-ligands relax young and aged murine cerebral arteries, whether this relates to the activation of PKA or Epac signaling pathways and is changed with advanced age. The hormone Urocortin-1 relaxed submaximally contracted young and old basilar arteries with a similar pD2 and DMAX (~ -8.5 and ~ 90% in both groups). In permeabilized arteries, PKA activation by 6-Bnz-cAMP or Epac activation by 8-pCPT-2'- O-Me-cAMP also induced relaxation with pD2 of -6.3 vs. -5.8 in old for PKA-ligands, and -4.4 and -4.0 in old for Epac-ligands. Furthermore, aging significantly increased submaximal Ca2+-induced force. The effect of 8-pCPT-2'-O-Me-cAMP on intact arteries was attenuated by aging or nitric oxide synthase inhibition. No relaxing effect in both age-groups was observed after treatment with PKAactivator, Sp-6-Phe-cAMPS. In conclusion, our results suggest that in intact basilar arteries relaxation induced by cAMP-mimetics refers only to the activation of Epac and is impaired by smooth muscle and endothelial aging. The study presents an interesting option allowing therapeutic discrimination between both pathways, possibly for the exclusive activation of Epac in brain circulatory system.

Role of endothelin-1 for the regulation of renal pelvic function.

Endothelin-1 (ET-1) stimulates contractions in isolated rat renal pelves. The signal transduction mechanisms that mediate ET-1-induced renal pelvic contractions and the role of ET-1 for the in vivo regulation of renal pelvic function are not well characterized. We tested if ET-1 stimulates contractions in murine and human renal pelves, if ET-1 activates the renal pelvic RhoA/ROCK pathway, and if low renal ET-1 formation or ET receptor blockade reduce renal pelvic contractile activity. ET-1 increased contraction frequency and force in murine renal pelves. The majority of human renal pelvic tissue samples showed tonic contractions in response to ET-1. Seven out of 20 human tissue samples showed phasic contractions. In four samples, they were elicited by ET-1 at 10-33 nmol/l. ET-1 increased renal pelvic RhoA-GTP content and myosin phosphatase target subunit 1 phosphorylation in isolated rat renal pelves. Renal pelvic contraction frequency (29 ± 2 vs. 29 ± 3 min(-1)) and renal pelvic pressure (7.1 ± 0.9 vs. 5.9 ± 1.7 mmHg) were similar in collecting duct-specific ET-1 knockout mice and in ET-1 floxed controls in vivo. ET-1 sensitivity of isolated renal pelves was similar in both groups. ET receptor blockade did not significantly affect pelvic contraction frequency and pressure in rats. We conclude that ET-1 stimulates phasic contractions in murine, rat, and, to a lesser extent, in human renal pelves. ET-1 activates the RhoA/ROCK pathway in the renal pelvic wall. Endogenous, kidney-derived ET-1 does not play a major role for the regulation of renal pelvic contractions in vivo.

OPN deficiency results in severe glomerulosclerosis in uninephrectomized mice.

Osteopontin (OPN) expression has been reported to be elevated in experimental models of renal injury such as arterial hypertension or diabetic nephropathy finally leading to focal segmental glomerulosclerosis (FSGS). FSGS is characterized by glomerular matrix deposition and loss or damage of podocytes that represent the main constituents of the glomerular filtration barrier. To evaluate the role of OPN in the kidney we investigated WT and OPN knockout mice (OPN-/-) without treatment, after uninephrectomy (UNX), as well as after UNX and desoxycorticosterone acetate (DOCA)-salt treatment with respect to urine parameters, glomerular morphology, and expression of podocyte markers. OPN-/- mice showed normal urine parameters while a thickening of the glomerular basement membrane was evident. Intriguingly, following UNX, OPN-/- mice exhibited prominent FSGS, proteinuria, and glomerular matrix deposition. Electron microscopy revealed bulgings of the glomerular basement membrane and occasionally an effacement of podocytes. After UNX and DOCA-salt treatment, severe glomerular lesions as well as proteinuria and albuminuria were seen in WT and OPN-/- mice. Moreover, we found a reduction of specific markers such as Wilm's tumor-1, podocin, and synaptopodin in both experimental groups indicating a loss of podocytes. Podocyte damage was accompanied by increased number of Ki-67-positive cells in the parietal epithelium of Bowman's capsule. We conclude that OPN plays a crucial role in adaptation of podocytes following renal ablation and is renoprotective when glomerular mechanical load is increased.

Comparison between cold plasma, electrochemotherapy and combined therapy in a melanoma mouse model.

The study was undertaken to compare antitumor efficacy of electrochemotherapy (ECT) with cold plasma therapy (CP) in a melanoma mouse model. After melanoma implantation into the flank of C57BL/6N mice, CP by two different plasma sources (APPJ and DBD) was applied directly to the tumor surface. ECT was performed with bleomycin intravenously at a field strength of 1000 V/cm without or combined with CP. Primary endpoints were tumor growth acceleration (TGA), daily volume progression (DVP) and survival after treatment. Both plasma sources as single treatment showed a significant TGA delay, which proved less effective than ECT. CP (APPJ) combined with ECT (ECJ) significantly improved per cent mouse survival, with significant superiority compared with ECT. Plasma therapy alone albeit less effective seems a potential alternative to ECT in patients with melanoma and can be applied manifold in a session without general anaesthesia. Accordingly, CP alone and combined with ECT may serve as new option in palliative skin melanoma therapy.

Potential benefits of rho-kinase inhibition in arterial hypertension.

Arterial hypertension is a major health problem, accounting for 12 % of the global death rate. A large proportion of patients treated for high blood pressure do not reach target blood pressure values. The question arises if new antihypertensive drugs could improve present hypertension treatment. Rho-kinases (ROCKs) are ubiquitously expressed serine/threonine kinases and involved in a variety of cell functions. They contribute to the pathogenesis of human and experimental hypertension. Pharmacological ROCK inhibition has been shown to effectively lower blood pressure in patients and experimental animals. Progress has been made towards the understanding on how non-selective ROCK inhibitors lower arterial pressure and efforts are currently undertaken to develop ROCK inhibitors to improve their specificity and isoenzyme selectivity. If introduction of ROCK inhibitors for the treatment of high blood pressure can significantly advance currently available options of antihypertensive pharmacotherapy awaits further experimental and clinical research.

Leptin potentiates endothelium-dependent relaxation by inducing endothelial expression of neuronal NO synthase.

OBJECTIVE: Obesity is associated with hyperleptinemia but it is not clear whether leptin protects vascular function or promotes dysfunction. We therefore studied the consequences of hyperleptinemia in lean mice. METHODS AND RESULTS: Wild-type and endothelial NO synthase (eNOS)(-/-) mice were infused with leptin (0.4 mg/kg per day, 7 days), and endothelium-dependent relaxation was studied in aortic segments. Leptin had no effect on acetylcholine-induced endothelium-dependent relaxation in normal wild-type mice but restored endothelium-dependent relaxation in wild-type mice treated with angiotensin II (0.7 mg/kg per day, 7 days) to induce endothelial dysfunction. Leptin also sensitized aortae from eNOS(-/-) mice to acetylcholine, an effect blocked by neuronal NOS (nNOS) inhibition and not observed in eNOS-nNOS double(-/-) mice. Consistent with these findings, leptin induced nNOS expression in murine and human vessels and human endothelial but not smooth muscle cells. Aortic nNOS expression was also induced in mice by a high-fat diet. Mechanistically, leptin increased endothelial Janus kinase 2 and signal transducer and activator of transcription 3 phosphorylation, and inhibition of Janus kinase 2 prevented nNOS induction in cultured cells and leptin-induced relaxations in eNOS(-/-) mice. CONCLUSIONS: Leptin induces endothelial nNOS expression, which compensates, in part, for a lack of NO production by eNOS to maintain endothelium-dependent relaxation.

Kv2.1 Channels Prevent Vasomotion and Safeguard Myogenic Reactivity in Rat Small Superior Cerebellar Arteries.

Vascular smooth muscle voltage-gated potassium (Kv) channels have been proposed to contribute to myogenic autoregulation. Surprisingly, in initial experiments, we observed that the Kv2 channel inhibitor stromatoxin induced vasomotion without affecting myogenic tone. Thus, we tested the hypothesis that Kv2 channels contribute to myogenic autoregulation by fine-tuning the myogenic response. Expression of Kv2 channel mRNA was determined using real-time PCR and 'multiplex' single-cell RT-PCR. Potassium currents were measured using the patch-clamp technique. Contractile responses of intact arteries were studied using isobaric myography. Expression of Kv2.1 but not Kv2.2 channels was detected in intact rat superior cerebellar arteries and in single smooth muscle cells. Stromatoxin, a high-affinity inhibitor of Kv2 channels, reduced smooth muscle Kv currents by 61% at saturating concentrations (EC50 36 nmol/L). Further, stromatoxin (10-100 nmol/L) induced pronounced vasomotion in 48% of the vessels studied. In vessels not exhibiting vasomotion, stromatoxin did not affect myogenic reactivity. Notably, in vessels exhibiting stromatoxin-induced vasomotion, pressure increases evoked two effects: First, they facilitated the occurrence of random vasodilations and/or vasoconstrictions, disturbing the myogenic response (24% of the vessels). Second, they modified the vasomotion by decreasing its amplitude and increasing its frequency, thereby destabilizing myogenic tone (76% of the vessels). Our study demonstrates that (i) Kv2.1 channels are the predominantly expressed Kv channels in smooth muscle cells of rat superior cerebellar arteries, and (ii) Kv2.1 channels provide a novel type of negative feedback mechanism in myogenic autoregulation by preventing vasomotion and thereby safeguarding the myogenic response.

ROK and RSK2-kinase pathways differ between senescent human renal and mesenteric arteries.

OBJECTIVE: Small arteries from different organs vary with regard to the mechanisms that regulate vasoconstriction. This study investigated the impact of advanced age on the regulation of vasoconstriction in isolated human small arteries from kidney cortex and periintestinal mesenteric tissue. METHODS: Renal and mesenteric tissues were obtained from patients (mean age 71 ±â€Š9 years) undergoing elective surgery. Furthermore, intrarenal and mesenteric arteries from young and aged mice were studied. Arteries were investigated by small vessel myography and western blot. RESULTS: Human intrarenal arteries (h-RA) showed higher stretch-induced tone and higher reactivity to α 1 adrenergic receptor stimulation than human mesenteric arteries (h-MA). Rho-kinase (ROK) inhibition resulted in a greater decrease in Ca 2+ and depolarization-induced tone in h-RA than in h-MA. Basal and α 1 adrenergic receptor stimulation-induced phosphorylation of the regulatory light chain of myosin (MLC 20 ) was higher in h-RA than in h-MA. This was associated with higher ROK-dependent phosphorylation of the regulatory subunit of myosin light-chain-phosphatase (MLCP), MYPT1-T853. In h-RA phosphorylation of ribosomal S6-kinase II (RSK2-S227) was significantly higher than in h-MA. Stretch-induced tone and RSK2 phosphorylation was also higher in interlobar arteries (m-IAs) from aged mice than in respective vessels from young mice and in murine mesenteric arteries (m-MA) from both age groups. CONCLUSION: Vasoconstriction in human intrarenal arteries shows a greater ROK-dependence than in mesenteric arteries. Activation of RSK2 may contribute to intrarenal artery tone dysregulation associated with aging. Compared with h-RA, h-MA undergo age-related remodeling leading to a reduction of the contractile response to α 1 adrenergic stimulation.

Oxidative Stress, Reductive Stress and Antioxidants in Vascular Pathogenesis and Aging.

This review is focused on the mechanisms that regulate health, disease and aging redox status, the signal pathways that counteract oxidative and reductive stress, the role of food components and additives with antioxidant properties (curcumin, polyphenols, vitamins, carotenoids, flavonoids, etc.), and the role of the hormones irisin and melatonin in the redox homeostasis of animal and human cells. The correlations between the deviation from optimal redox conditions and inflammation, allergic, aging and autoimmune responses are discussed. Special attention is given to the vascular system, kidney, liver and brain oxidative stress processes. The role of hydrogen peroxide as an intracellular and paracrine signal molecule is also reviewed. The cyanotoxins ß-N-methylamino-l-alanine (BMAA), cylindrospermopsin, microcystins and nodularins are introduced as potentially dangerous food and environment pro-oxidants.

Leukocyte telomere length and mitochondrial DNA copy number associate with endothelial function in aging-related cardiovascular disease.

Background: We investigated the association between leukocyte telomere length, mitochondrial DNA copy number, and endothelial function in patients with aging-related cardiovascular disease (CVD). Methods: In total 430 patients with CVD and healthy persons were enrolled in the current study. Peripheral blood was drawn by routine venipuncture procedure. Plasma and peripheral blood mononuclear cells (PBMCs) were collected. Cell-free genomic DNA (cfDNA) and leukocytic genomic DNA (leuDNA) were extracted from plasma and PBMCs, respectively. Relative telomere length (TL) and mitochondrial DNA copy number (mtDNA-CN) were analyzed using quantitative polymerase chain reaction. Endothelial function was evaluated by measuring flow-mediated dilation (FMD). The correlation between TL of cfDNA (cf-TL), mtDNA-CN of cfDNA (cf-mtDNA), TL of leuDNA (leu-TL), mtDNA-CN of leuDNA (leu-mtDNA), age, and FMD were analyzed based on Spearman's rank correlation. The association between cf-TL, cf-mtDNA, leu-TL, leu-mtDNA, age, gender, and FMD were explored using multiple linear regression analysis. Results: cf-TL positively correlated with cf-mtDNA (r = 0.1834, P = 0.0273), and leu-TL positively correlated with leu-mtDNA (r = 0.1244, P = 0.0109). In addition, both leu-TL (r = 0.1489, P = 0.0022) and leu-mtDNA (r = 0.1929, P < 0.0001) positively correlated with FMD. In a multiple linear regression analysis model, both leu-TL (ß = 0.229, P = 0.002) and leu-mtDNA (ß = 0.198, P = 0.008) were positively associated with FMD. In contrast, age was inversely associated with FMD (ß = -0.426, P < 0.0001). Conclusion: TL positively correlates mtDNA-CN in both cfDNA and leuDNA. leu-TL and leu-mtDNA can be regarded as novel biomarkers of endothelial dysfunction.

Dual thick and thin filament linked regulation of stretch- and L-NAME-induced tone in young and senescent murine basilar artery.

Stretch-induced vascular tone is an important element of autoregulatory adaptation of cerebral vasculature to maintain cerebral flow constant despite changes in perfusion pressure. Little is known as to the regulation of tone in senescent basilar arteries. We tested the hypothesis, that thin filament mechanisms in addition to smooth muscle myosin-II regulatory-light-chain-(MLC20)-phosphorylation and non-muscle-myosin-II, contribute to regulation of stretch-induced tone. In young BAs (y-BAs) mechanical stretch does not lead to spontaneous tone generation. Stretch-induced tone in y-BAs appeared only after inhibition of NO-release by L-NAME and was fully prevented by treatment with 3 µmol/L RhoA-kinase (ROK) inhibitor Y27632. L-NAME-induced tone was reduced in y-BAs from heterozygous mice carrying a point mutation of the targeting-subunit of the myosin phosphatase, MYPT1 at threonine696 (MYPT1-T696A/+). In y-BAs, MYPT1-T696A-mutation also blunted the ability of L-NAME to increase MLC20-phosphorylation. In contrast, senescent BAs (s-BAs; >24 months) developed stable spontaneous stretch-induced tone and pharmacological inhibition of NO-release by L-NAME led to an additive effect. In s-BAs the MYPT1-T696A mutation also blunted MLC20-phosphorylation, but did not prevent development of stretch-induced tone. In s-BAs from both lines, Y27632 completely abolished stretch- and L-NAME-induced tone. In s-BAs phosphorylation of non-muscle-myosin-S1943 and PAK1-T423, shown to be down-stream effectors of ROK was also reduced by Y27632 treatment. Stretch- and L-NAME tone were inhibited by inhibition of non-muscle myosin (NM-myosin) by blebbistatin. We also tested whether the substrate of PAK1 the thin-filament associated protein, caldesmon is involved in the regulation of stretch-induced tone in advanced age. BAs obtained from heterozygotes Cald1+/- mice generated stretch-induced tone already at an age of 20-21 months old BAs (o-BA). The magnitude of stretch-induced tone in Cald1+/- o-BAs was similar to that in s-BA. In addition, truncation of caldesmon myosin binding Exon2 (CaD-▵Ex2-/-) did not accelerate stretch-induced tone. Our study indicates that in senescent cerebral vessels, mechanisms distinct from MLC20 phosphorylation contribute to regulation of tone in the absence of a contractile agonist. While in y-and o-BA the canonical pathways, i.e., inhibition of MLCP by ROK and increase in pMLC20, predominate, tone regulation in senescence involves ROK regulated mechanisms, involving non-muscle-myosin and thin filament linked mechanisms involving caldesmon.

Kidney-specific deletion of multidrug resistance-related protein 2 does not aggravate acute cyclosporine A nephrotoxicity in rats.

OBJECTIVE: Multidrug resistance-related protein 2 (Mrp2) is expressed in apical membranes of renal proximal tubular cells and contributes to the renal secretion of cyclosporine A (CsA). Mrp2⁻/⁻ deficiency may lead to local renal CsA accumulation. We investigated whether kidney-specific Mrp2 deficiency enhances acute CsA nephrotoxicity in rats. METHODS: Kidney-specific Mrp2 deletion was achieved by bilateral nephrectomy and transplantation of a congenic Mrp2-deficient kidney into wild-type recipients. Controls received a wild-type kidney. Animals were treated with CsA (10 or 30 mg/kg/day) for 7 days. Renal hemodynamics and renal cortical mRNA expression profile, oxidative stress, and the abundance of multidrug resistance protein 1 (Mdr1) and Mrp2 were assessed. RESULTS: CsA accumulation and CsA-induced reduction in glomerular filtration rate were similar in wild-type and Mrp2⁻/⁻ kidneys. Renal vascular resistance and agonist-induced renal vascular responses were similar in both groups. A PCR array on 84 genes involved in the biotransformation and antioxidant defense revealed increased CsA-induced mRNA expression of genes involved in oxidative and metabolic stress, inflammation, and apoptosis. This gene expression pattern was similar in wild-type and Mrp2⁻/⁻ kidneys. CsA increased the renal cortical oxidized glutathione, did not affect xanthine oxidase-dependent superoxide formation, and decreased renal cortical NADPH oxidase-dependent superoxide formation. Furthermore, CsA increased Mdr1 protein abundance to a greater extent in Mrp2⁻/⁻ than in wild-type kidneys. CONCLUSION: Mrp2 is not critical for renal CsA disposition and its deficiency does not enhance acute CsA nephrotoxicity. The high Mdr1 abundance may at least in part prevent exaggerated CsA accumulation in Mrp2⁻/⁻ kidneys.

Senescent murine femoral arteries undergo vascular remodelling associated with accelerated stress-induced contractility and reactivity to nitric oxide.

This work explored the mechanism of augmented stress-induced vascular reactivity of senescent murine femoral arteries (FAs). Mechanical and pharmacological reactivity of young (12-25 weeks, y-FA) and senescent (>104 weeks, s-FAs) femoral arteries was measured by wire myography. Expression and protein phosphorylation of selected regulatory proteins were studied by western blotting. Expression ratio of the Exon24 in/out splice isoforms of the regulatory subunit of myosin phosphatase, MYPT1 (MYPT1-Exon24 in/out), was determined by polymerase chain reaction (PCR). While the resting length-tension relationship showed no alteration, the stretch-induced-tone increased to 8.3 ± 0.9 mN in s-FA versus only 4.6 ± 0.3 mN in y-FAs. Under basal conditions, phosphorylation of the regulatory light chain of myosin at S19 was 19.2 ± 5.8% in y-FA versus 49.2 ± 12.6% in s-FA. Inhibition of endogenous NO release raised tone additionally to 10.4 ± 1.2 mN in s-FA, whereas this treatment had a negligible effect in y-FAs (4.8 ± 0.3 mN). In s-FAs, reactivity to NO donor was augmented (pD2  = -4.5 ± 0.3 in y-FA vs. -5.2 ± 0.1 in senescent). Accordingly, in s-FAs, MYPT1-Exon24-out-mRNA, which is responsible for expression of the more sensitive to protein-kinase G, leucine-zipper-positive MYPT1 isoform, was increased. The present work provides evidence that senescent murine s-FA undergoes vascular remodelling associated with increases in stretch-activated contractility and sensitivity to NO/cGMP/PKG system.

Angiotensin receptor-neprilysin inhibitor improves coronary collateral perfusion.

Background: We investigated the pleiotropic effects of an angiotensin receptor-neprilysin inhibitor (ARNi) on collateral-dependent myocardial perfusion in a rat model of coronary arteriogenesis, and performed comprehensive analyses to uncover the underlying molecular mechanisms. Methods: A rat model of coronary arteriogenesis was established by implanting an inflatable occluder on the left anterior descending coronary artery followed by a 7-day repetitive occlusion procedure (ROP). Coronary collateral perfusion was measured by using a myocardial particle infusion technique. The putative ARNi-induced pro-arteriogenic effects were further investigated and compared with an angiotensin-converting enzyme inhibitor (ACEi). Expression of the membrane receptors and key enzymes in the natriuretic peptide system (NPS), renin-angiotensin-aldosterone system (RAAS) and kallikrein-kinin system (KKS) were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblot assay, respectively. Protein levels of pro-arteriogenic cytokines were measured by enzyme-linked immunosorbent assay, and mitochondrial DNA copy number was assessed by qPCR due to their roles in arteriogenesis. Furthermore, murine heart endothelial cells (MHEC5-T) were treated with a neprilysin inhibitor (NEPi) alone, or in combination with bradykinin receptor antagonists. MHEC5-T proliferation was analyzed by colorimetric assay. Results: The in vivo study showed that ARNis markedly improved coronary collateral perfusion, regulated the gene expression of KKS, and increased the concentrations of relevant pro-arteriogenic cytokines. The in vitro study demonstrated that NEPis significantly promoted MHEC5-T proliferation, which was diminished by bradykinin receptor antagonists. Conclusion: ARNis improve coronary collateral perfusion and exert pro-arteriogenic effects via the bradykinin receptor signaling pathway.

Endothelin-1-induced activation of rat renal pelvic contractions depends on cyclooxygenase-1 and Rho kinase.

Upper urinary tract peristalsis is generated in the proximal renal pelvis that connects to the renal parenchyma at the pelvis-kidney junction. It may be exposed to the high renal endothelin-1 (ET-1) concentrations. Dietary NaCl restriction increases renal pelvic ET(A) receptor expression. We investigated the contribution of ET(A) and ET(B) receptors to ET-1-stimulated rat renal pelvic contractions and whether the sensitivity of renal pelvic contractile activity to ET-1 stimulation increases with dietary NaCl restriction. We tested whether ET-1-induced contractile activity depends on cyclooxygenase (COX)-1 or -2 and to what extent spontaneous as well as agonist-induced peristalsis depends on Rho kinases (ROCK). Contractions of isolated renal pelvises were investigated by myography. ET-1 concentration-dependently increased pelvic contractile activity up to 400% of basal activity. ET(A) but not ET(B) receptor blockade inhibited ET-1-induced pelvic contractions. Basal and ET-1-stimulated contractions were similar in renal pelvises from rats on a high-NaCl diet or on a NaCl-deficient diet. COX-1 inhibition reduced spontaneous and almost completely blocked the ET-1-induced pelvic contractions. ROCK inhibition reduced spontaneous and ET-1 stimulated pelvic contractile activity by 90%. RT-PCR revealed that both ROCK isoenzymes are present in the renal pelvic wall. Western blot analyses did not show increased phosphorylation of ROCK substrates myosin phosphatase target subunit 1, ezrin, radixin, and moesin in ET-1-treated isolated renal pelvises. ET-1 is a powerful ET(A) receptor-dependent activator of renal pelvic contractions. COX-1 and ROCK activity are required for the ET-1 effects on pelvic contractions, which are not significantly affected by dietary NaCl intake.

The sympathetic nervous system in acute kidney injury.

Acute kidney injury (AKI) is frequently accompanied by activation of the sympathetic nervous system (SNS). This may result from pre-exisiting chronic diseases associated with sympathetic activation prior to AKI or it may be induced by stressors that ultimately lead to AKI such as endotoxins and arterial hypotension in circulatory shock. Conversely, sympathetic activation may also result from acute renal injury. Focusing on studies in experimental renal ischaemia and reperfusion (IR), this review summarizes the current knowledge on how the SNS is activated in IR-induced AKI and on the consequences of sympathetic activation for the development of acute renal damage. Experimental studies show beneficial effects of sympathoinhibitory interventions on renal structure and function in response to IR. However, few clinical trials obtained in scenarios that correspond to experimental IR, namely major elective surgery, showed that peri-operative treatment with centrally acting sympatholytics reduced the incidence of AKI. Apparently, discrepant findings on how sympathetic activation influences renal responses to acute IR-induced injury are discussed and future areas of research in this field are identified.

Dietary sodium modulates the interaction between efferent renal sympathetic nerve activity and afferent renal nerve activity: role of endothelin.

Increasing efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA), which in turn decreases ERSNA via activation of the renorenal reflexes in the overall goal of maintaining low ERSNA. We now examined whether the ERSNA-induced increases in ARNA are modulated by dietary sodium and the role of endothelin (ET). The ARNA response to reflex increases in ERSNA was enhanced in high (HNa)- vs. low-sodium (LNa) diet rats, 7,560 +/- 1,470 vs. 900 +/- 390%.s. The norepinephrine (NE) concentration required to increase PGE(2) and substance P release from isolated renal pelvises was 10 pM in HNa and 6,250 pM in LNa diet rats. In HNa diet pelvises 10 pM NE increased PGE(2) release from 67 +/- 6 to 150 +/- 13 pg/min and substance P release from 6.7 +/- 0.8 to 12.3 +/- 1.8 pg/min. In LNa diet pelvises 6,250 pM NE increased PGE(2) release from 64 +/- 5 to 129 +/- 22 pg/min and substance P release from 4.5 +/- 0.4 to 6.6 +/- 0.7 pg/min. In the renal pelvic wall, ETB-R are present on unmyelinated Schwann cells close to the afferent nerves and ETA-R on smooth muscle cells. ETA-receptor (R) protein expression in the renal pelvic wall is increased in LNa diet. In HNa diet, renal pelvic administration of the ETB-R antagonist BQ788 reduced ERSNA-induced increases in ARNA and NE-induced release of PGE(2) and substance P. In LNa diet, the ETA-R antagonist BQ123 enhanced ERSNA-induced increases in ARNA and NE-induced release of substance P without altering PGE(2) release. In conclusion, activation of ETB-R and ETA-R contributes to the enhanced and suppressed interaction between ERSNA and ARNA in conditions of HNa and LNa diet, respectively, suggesting a role for ET in the renal control of ERSNA that is dependent on dietary sodium.

Apocynin-induced vasodilation involves Rho kinase inhibition but not NADPH oxidase inhibition.

AIMS: The present study was designed to test the hypothesis that NADPH oxidase inhibition with apocynin would lower blood pressure and improve endothelial function in spontaneously hypertensive rats (SHRs). Although apocyin effectively dilated arterial segments in vitro, it failed to lower blood pressure or improve endothelial function. Further experiments were performed in normotensive rats and in NADPH oxidase subunit knock-out mice to test if apocynin-induced vasodilation depends on NADPH oxidase inhibition at all. METHODS AND RESULTS: SHRs were treated with apocynin orally or i.v. Arterial pressure was recorded directly. Rat and mouse arterial function was investigated in vitro by small vessel wire myography. NADPH oxidase activity was measured in human granulocytes and in rat vascular preparations. Rho kinase activity was determined by Western blot analysis. Apocynin did not reduce arterial pressure acutely in SHR when given at 50, 100, or 150 mg kg(-1) day(-1) orally over 1-week intervals or when given i.v. Apocynin potently inhibited granulocyte NADPH oxidase but not vascular NADPH-oxidase-dependent oxygen radical formation unless exogenous peroxidase was added to vascular preparations. Apocynin dilated rat intrarenal and coronary arteries independently of pharmacological interventions that reduce vascular superoxide radical abundance and actions. Aortic rings from p47phox(-/-) mice were more sensitive to apocynin-induced dilation than wild-type aortic rings. Rho kinase inhibition reduced or prevented the inhibitory effect of apocynin on agonist-induced vasoconstriction and apocynin inhibited the phosphorylation of Rho kinase substrates. CONCLUSION: Apocynin per se does not inhibit vascular NADPH-oxidase-dependent superoxide formation. Its in vitro vasodilator actions are not due to NADPH oxidase inhibition but may be explained at least in part by inhibition of Rho kinase activity. The discrepancy between apocynin-induced vasodilation in vitro and the failure of apocynin to lower arterial pressure in SHR suggests opposing effects on arterial pressure-regulating systems in vivo. Its use as a pharmacological tool to investigate vascular NADPH oxidase should be discontinued.