Extravasal albumin concentration modulates contractile responses of renal afferent arterioles.

AIM: Afferent arterioles (AA) hold a key position in the regulation of renal blood flow and glomerular filtration rate. Being the effector site of tubuloglomerular feedback, the afferent arteriole contributes to the renal handling of sodium and fluid. Dehydration goes along with increased renal interstitial protein concentration. Here, the hypothesis was tested that extravasal protein concentration directly modulates afferent arteriolar tone, a mechanism which may contribute to body fluid volume control. METHOD: The effect of increased extravasal albumin concentration on the vascular reactivity was investigated in renal AA and interlobar arteries of mice, in rat renal AA and in pancreatic islet arterioles. RESULTS: Albumin (2 and 4% in the bath solution) significantly potentiated the contractile response of renal afferent arterioles induced by angiotensin II and adenosine, as well as their combination, compared to the control situation (0.1% albumin). Albumin did not influence the contractility of larger renal vessels or pancreatic islet arterioles. Mimicking the increase in the osmolality induced by 4% albumin by applying mannitol to the bath solution also increased the response of renal arterioles to Ang II. However, the effect was smaller compared to that of albumin. The nitric oxide bioavailability, measured by DAF-FM fluorescence, was reduced in afferent arterioles exposed to 4% albumin. CONCLUSION: The protein-induced modulation of AA tone is mediated by the increased osmolality as well as by NO scavenging. The results suggest a possible contribution of these mechanisms to the control of extracellular fluid volume via adjustment of renal blood flow and glomerular filtration rate.

Increased hydrogen peroxide impairs angiotensin II contractions of afferent arterioles in mice after renal ischaemia-reperfusion injury.

AIM: Renal ischaemia-reperfusion injury (IRI) increases angiotensin II (Ang II) and reactive oxygen species (ROS) that are potent modulators of vascular function. However, the roles of individual ROS and their interaction with Ang II are not clear. Here we tested the hypothesis that IRI modulates renal afferent arteriolar responses to Ang II via increasing superoxide (O2-) or hydrogen peroxide (H2 O2 ). METHODS: Renal afferent arterioles were isolated and perfused from C57BL/6 mice 24 h after IRI or sham surgery. Responses to Ang II or noradrenaline were assessed by measuring arteriolar diameter. Production of H2 O2 and O2- was assessed in afferent arterioles and renal cortex. Activity of SOD and catalase, and mRNA expressions of Ang II receptors were assessed in pre-glomerular arterioles and renal cortex. RESULTS: Afferent arterioles from mice after IRI had a reduced maximal contraction to Ang II (-27±2 vs. -42±1%, P < 0.001), but retained a normal contraction to noradrenaline. Arterioles after IRI had a 38% increase in H2 O2 (P < 0.001) and a 45% decrease in catalase activity (P < 0.01). Contractions were reduced in normal arterioles after incubation with H2 O2 (-22±2 vs. -42±1%, P < 0.05) similar to the effects of IRI. However, the impaired contractions were normalized by incubation with PEG catalase despite a reduced AT1 R expression. CONCLUSIONS: Renal IRI in mice selectively impairs afferent arteriolar responses to Ang II because of H2 O2 accumulation that is caused by a reduced catalase activity. This could serve to buffer the effect of Ang II after IRI and may be a protective mechanism.

Diadenosine pentaphosphate modulates glomerular arteriolar tone and glomerular filtration rate.

INTRODUCTION: Mechanisms and participating substances involved in the reduction of glomerular filtration (GFR) in contrast-induced acute kidney injury (CI-AKI) are still matter of debate. We hypothesized that diadenosine polyphosphates are released by the action of contrast media on tubular cells and may act on glomerular arterioles and reduce GFR. METHODS: Freshly isolated rat tubules were treated with the contrast medium iodixanol (47 mg iodine per mL) at 37 °C for 20 min. The content of Apn A (n = 3-6) in the supernatant of treated tubules and in the plasma of healthy persons and patients with AKI was analysed using reversed-phase chromatography, affinity chromatography and mass spectrometry. GFR was obtained in conscious mice by inulin clearance. Concentration response curves for Apn A (n = 3-6, 10(-12) -10(-5)  mol L(-1) ) were measured in isolated perfused glomerular arterioles. RESULTS: Iodixanol treatment of tubules significantly increased the concentration of Apn A (n = 3-5) in the supernatant. Ap6 A was below the detection limit. AKI patient shows higher concentrations of Apn A compared to healthy. Application of Ap5 A significantly reduced the GFR in conscious mice. Ap5 A reduced afferent arteriolar diameters, but did not influence efferent arterioles. The constrictor effect on afferent arterioles was strong immediately after application, but weakened with time. Then, non-selective P2 inhibitor suramin blocked the Ap5 A-induced constriction. CONCLUSION: The data suggest that Ap5 A plays a role in the pathophysiology of CI-AKI. We show a contrast media-induced release of Ap5 A from tubules, which might increase afferent arteriolar resistance and reduce the GFR.

Adenosine A1 receptor-dependent and independent pathways in modulating renal vascular responses to angiotensin II.

AIM: Renal afferent arterioles are the effector site for autoregulation of glomerular perfusion and filtration. There is synergistic interaction between angiotensin II (ANG II) and adenosine (Ado) in regulating arteriolar contraction; however, the mechanisms are not clear. In this context, this study investigated the contribution of A1 receptor-dependent and independent signalling mechanisms. METHODS: Isolated perfused afferent arterioles from transgenic mice (A1 (+/+) and A1 (-/-) ) were used for vascular reactivity studies. Cultured vascular smooth muscle cells (VSMC) were used for phosphorylation studies of signalling proteins that induce arteriolar contraction. RESULTS: Maximal arteriolar contraction to ANG II was attenuated in A1 (-/-) (22%) compared with A1 (+/+) (40%). Simultaneous incubation with low-dose ado (10(-8)  mol L(-1) ) enhanced ANG II-induced contraction in A1 (+/+) (58%), but also in A1 (-/-) (42%). An ado transporter inhibitor (NBTI) abolished this synergistic effect in A1 (-/-) , but not in wild-type mice. Incubation with Ado + ANG II increased p38 phosphorylation in aortic VSMC from both genotypes, but treatment with NBTI only blocked phosphorylation in A1 (-/-) . Combination of ANG II + Ado also increased MLC phosphorylation in A1 (+/+) but not significantly in A1 (-/-) , and NBTI had no effects. In agreement, Ado + ANG II-induced phosphorylation of p38 and MLC in rat pre-glomerular VSMC was not affected by NBTI. However, during pharmacological inhibition of the A1 receptor simultaneous treatment with NBTI reduced phosphorylation of both p38 and MLC to control levels. CONCLUSION: Interaction between ANG II and Ado in VSMC normally involves A1 receptor signalling, but this can be compensated by receptor independent actions that phosphorylate p38 MAPK and MLC.

Changes in arterial function in a mouse model of human familial hypercholesterolaemia.

AIM: Atherosclerosis is the most common cause of cardiovascular disease. The ApoB mouse is a model for human familial hypercholesterolaemia and has a lipoprotein profile similar to that of humans with atherosclerosis. Therefore, it is a suitable model to investigate the changes in vasoreactivity during atherogenesis. This study investigates contractile and dilatative properties of arteries in this model in relation to age. METHODS: Male ApoB mice and B6, wild-type (WT), mice were examined at age four or 18 months. Isometric measurements of 2-mm ring preparations of the aorta thoracica were performed using a wire myograph. Histological and biochemical methods served to determine atherosclerosis, lipid status and endothelial markers respectively. RESULTS: Morphometric analysis showed that all old ApoB mice had severe atherosclerosis in the aorta. Atherosclerotic alteration of the aorta of the ApoB mice coincided with a diminished vasodilatation to acetylcholine. The phenylephrine response was significantly attenuated already to the same degree in the non-atherosclerotic aorta of the young ApoB mice as in the atherosclerotic aorta of the older ApoB mice. Serum parameters showed a rise in total cholesterol and triglycerides in the ApoB strain compared to WT mice. Soluble intercellular adhesion molecule (sICAM)-1 and soluble vascular adhesion molecule (sVCAM)-1 were increased in old compared to young ApoB mice. CONCLUSION: The study shows that reduced acetylcholine-induced dilatation is related to the presence of atherosclerosis in old ApoB mice. Remarkably, the impaired vessel reactivity to phenylephrine already in young ApoB mice indicates early changes in vascular function in this model.

Functional characterization of isolated, perfused outermedullary descending human vasa recta.

AIM: The renal medulla plays an important role in the control of water and salt balance by the kidney. Outer medullary descending vasa recta (OMDVR) are microscopic vessels providing blood flow to the renal medulla. Data on the physiology of human vasa recta are scarce. Therefore, we established an experimental model of human single isolated, perfused OMDVR and characterized their vasoactivity in response to angiotensin II and to pressure changes. METHODS: Human non-malignant renal tissue was obtained from patients undergoing nephrectomy due to renal cell carcinoma. OMDVR were dissected under magnification and perfused using concentric microscopic pipettes. The response of OMDVR to angiotensin II and pressure changes was quantified in serial pictures. All patients signed a consent form prior to surgery. RESULTS: Outer medullary descending vasa recta constricted significantly after bolus applications of angiotensin II. OMDVR constriction to angiotensin II was also concentration dependent. Response to luminal pressure changes was different according to the diameter of vessels, with larger OMDVR constricting after pressure increase, while smaller ones did not. CONCLUSION: Outer medullary descending vasa recta constrict in response to angiotensin II and pressure increases. Our results show that OMDVR may take part in the regulation of medullary blood flow in humans. Our model may be suitable for investigating disturbances of renal medullary circulation in human subjects.

Angiotensin II enhances the afferent arteriolar response to adenosine through increases in cytosolic calcium.

AIMS: Angiotensin II (Ang II) is a strong renal vasoconstrictor and modulates the tubuloglomerular feedback (TGF). We hypothesized that Ang II at low concentrations enhances the vasoconstrictor effect of adenosine (Ado), the mediator of TGF. METHODS: Afferent arterioles of mice were isolated and perfused, and both isotonic contractions and cytosolic calcium transients were measured. RESULTS: Bolus application of Ang II (10(-12) and 10(-10) M) induced negligible vasoconstrictions, while Ang II at 10(-8) m reduced diameters by 35%. Ang II at 10(-12), 10(-10) and 10(-8) m clearly enhanced the arteriolar response to cumulative applications of Ado (10(-11) to 10(-4) M). Ado application increased the cytosolic calcium concentrations in the vascular smooth muscle, which were higher at 10(-5) M than at 10(-8) M. Ang II (10(-11) to 10(-6) M) also induced concentration-dependent calcium transients, which were attenuated by AT(1) receptor inhibition. Simultaneously applied Ang II (10(-10) M) additively enhanced the calcium transients induced by 10(-8) and 10(-5) M Ado. The transients were partly inhibited by AT(1) or A(1) receptor antagonists, but not significantly by A(2) receptor antagonists. CONCLUSION: A low dose of Ang II enhances Ado-induced constrictions, partly via AT(1) receptor-mediated calcium increase. Ado increases intracellular calcium by acting on A(1) but not A(2) receptors. The potentiating effect of Ang II on Ado-induced arteriolar vasoconstrictions may involve calcium sensitization of the contractile machinery, as Ang II only additively increased cytosolic calcium concentrations, while its effect on the arteriolar constriction was more than additive. The potentiating effect of Ang II might contribute to the resetting of TGF.

Uridine adenosine tetraphosphate acts as an autocrine hormone affecting glomerular filtration rate.

Recently, uridine adenosine tetraphosphate (Up(4)A) was described as a strong vasoconstrictor released from endothelial cells after stimulation with mechanical stress. In this study, we isolated and identified Up(4)A from kidney tissue, and we characterized the essential varying effects of Up(4)A on the afferent and efferent arterioles. Porcine and human kidney tissue was fractionated by size exclusion chromatography, affinity chromatography, anion exchange chromatography and reverse phase chromatography. In fractions purified to homogeneity, Up(4)A was identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), MALDI-LIFT fragment mass spectrometry (MALDI-TOF/TOF MS), retention-time comparison and enzymatic cleavage analysis. We analysed the release of Up(4)A from cultivated renal proximal tubule cells after stimulation of protein kinase C with oleoyl-2-acetyl-sn-glycerol (OAG). Up(4)A was identified in renal tissue, and the effect of Up(4)A on the vascular tone of isolated perfused afferent and efferent arterioles was tested. Stimulation of tubule cells with OAG increased the release rate of Up(4)A from tubule cells about tenfold. Up(4)A acts as a strong vasoconstrictive mediator on afferent arterioles, but has no significant effect on the tone of efferent arterioles, suggesting a functional role of Up(4)A as an autocrine hormone for glomerular perfusion. Because of the predominant effect of the Up(4)A on afferent arterioles, we assume that Up(4)A may decrease glomerular perfusion, intra-glomerular pressure and, hence, glomerular filtration rate. The release of Up(4)A from renal tubular cells may be an additional mechanism whereby tubular cells could affect renal perfusion. Up(4)A release may further contribute to renal vascular autoregulation mechanisms. In conclusion, as Up(4)A occurs in renal tissue and has marked effects on afferent but not efferent arterioles, Up(4)A may play a role in renal hemodynamics and possibly blood pressure regulation.

Adenosine increases calcium sensitivity via receptor-independent activation of the p38/MK2 pathway in mesenteric arteries.

AIM: Adenosine (Ado) restores desensitized angiotensin II-induced contractions in the renal arterioles via an intracellular, receptor-independent mechanisms including the p38 mitogen-activated protein kinase (MAPK). In the present study we test the hypothesis that MAPK-activated protein kinase 2 (MK2) mediates the Ado effect downstream from p38 MAPK resulting in an increased phosphorylation of the regulatory unit of the myosin light chain (MLC(20)). METHODS AND RESULTS: Contraction experiments were performed in rings of mesenteric arteries under isometric conditions in C57BL6 and MK2 knock out mice (MK2-/-). Ado pretreatment (10(-5) mol L(-1)) strongly increased Ang II sensitivity, calcium sensitivity and the phosphorylation of MLC(20). Treatment with Ado (3 x 10(-6) or 10(-5) mol L(-1) in between successive Ang II applications) enhanced the desensitized Ang II responses (second to fifth application). Ca(2+) transients were not effected by Ado. Further, blockade of type 1 and type 2 Ado receptors during treatment did not influence the effect. Type 3 receptor activation by inosine instead of Ado had no effect. Conversely, inhibition of nitrobenzylthioinosine-sensitive Ado transporters prevented the effects of Ado. Inhibition of p38 MAPK as well as use of MK2-/- mice prevented contractile Ado effects on the mesenteric arteries and the phosphorylation of MLC(20). CONCLUSION: The study shows that Ado activates the p38 MAPK/MK2 pathway in vascular smooth muscle via an intracellular action, which results in an increased MLC(20) phosphorylation in concert with increased calcium sensitivity of the contractile apparatus. This mechanism can significantly contribute to the regulation of vascular tone, e.g. under post-ischaemic conditions.

Contribution of adenosine receptors in the control of arteriolar tone and adenosine-angiotensin II interaction.

Adenosine (Ado) mediates vasoconstriction via A(1)-Ado receptors and vasodilation via A(2)-Ado receptors in the kidney. It interacts with angiotensin II (Ang II), which is important for renal hemodynamics and tubuloglomerular feedback (TGF). The aim was to investigate the function of Ado receptors in the Ado-Ang II interaction in mouse microperfused, afferent arterioles. Ado (10(-11)-10(-4) mol/l) caused a biphasic response: arteriolar diameters were reduced (-7%) at Ado 10(-11)-10(-9) mol/l and returned to control values at higher concentrations. Treatment with Ang II (10(-10) mol/l) transformed the response into a concentration-dependent constriction. N(6)-cyclopentyladenosine (A(1)-Ado receptor agonist) reduced diameters (12% at 10(-6) mol/l). Application of CGS21680 (10(-12)-10(-4) mol/l, A(2A) receptor agonist) increased the diameter by 13%. Pretreatment with ZM241385 (A(2A)-Ado receptor antagonist) alone or in combination with MRS1706 (A(2B)-Ado receptor antagonist) resulted in a pure constriction upon Ado, whereas 8-cyclopentyltheophylline (CPT) (A(1)-Ado receptor antagonist) inhibited the constrictor response. Afferent arterioles of mice lacking A(1)-Ado receptor did not show constriction upon Ado. Treatment with Ado (10(-8) mol/l) increased the response upon Ang II, which was blocked by CPT. Ado (10(-5) mol/l) did not influence the Ang II response, but an additional blockade of A(2)-Ado receptors enhanced it. The action of Ado on constrictor A(1)-Ado receptors and dilatory A(2)-Ado receptors modulates the interaction with Ang II. Both directions of Ado-Ang II interaction, which predominantly leads to an amplification of the contractile response, are important for the operation of the TGF.

Time of measurement influences the variability of tidal breathing parameters in healthy and sick infants.

The aim of this study was to investigate the influence of the time, when measuring tidal breathing parameters 1 min (epoch 1) and 5 min (epoch 2) after application of the facemask in healthy infants and infants with bronchopulmonary dysplasia (BPD), using the dead space free flow-through technique. In both patient groups, there were no statistically significant differences between epoch 1 and 2, in most of the tidal breathing parameters, except an increased VE and increased correlation dimension of the respiratory signal in the BPD infants in epoch 1. However, in nearly all parameters the coefficient of variation (CV) was significantly higher in epoch 1 compared with epoch 2, and in some infants, we found very high CVs (>50%) in epoch 1, which disappeared in epoch 2. The study shows that after having applied the facemask, a sufficient amount of adaptation time is necessary in order to reduce the within-subject variability and improve the reproducibility and interpretation of tidal breathing measurements in infants.

Rhythms and complexity of respiration during sleep in pre-term infants.

The aim of this study is to test rhythmic and complex properties of respiratory control in former ventilated, pre-term infants during quiet and active sleep. The children had a higher risk for sudden infant death due to bronchopulmonary dysplasia (BPD). Twelve infants suffering from BPD and 12 control infants, matched regarding their post-conceptional age, were examined polygraphically during quiet (QS) and active sleep (AS). The respiratory rate (RR), the ratio (LF/HF) between the low-frequency power (LF) and the high-frequency power (HF) of the spectra of the thoracic respiratory effort, and the frequency of the dominant peak within LF (LFF) and HF (HFF) were computed. The correlation dimension (D2) of the respiratory signal was calculated to determine the complexity of the respiratory control. The transcutaneous pO2 (tcpO2) and pCO2 and the oxygen saturation (sO2) were analysed. Infants with BPD had significantly higher RR and HFF during QS (median: BPD 48 breaths min-1; control 32 breaths min-1). tcpO2 and sO2 were significantly lower in the BPD group. No differences were found in LF/HF, LFF or D2 between groups, either in QS or in AS. D2 ranged between 1.8 and 3.8, showing significantly higher values during AS. LFF was found to be lower during active sleep (AS 0.04-0.05 Hz; QS about 0.06 Hz). We propose that in infants with BPD the lower lung compliance and the higher resistance, and possibly also the hypoxaemia, contribute to the acceleration of breathing. The behaviour of RR, spectral parameters and D2 indicates a specific, functional setting rather than a regulatory impairment in infants with BPD.

Linear and nonlinear properties of heart rate control in infants at risk.

The aim of this study was to test whether the heart rate (HR) control in infants at risk differs in comparison with healthy infants. Twelve former preterm infants suffering from bronchopulmonary dysplasia and 18 control infants, matched for their postconceptional age, were examined polygraphically during quiet and active sleep. HR, low-frequency (LF) power, high-frequency (HF) power, total power, and the ratio of LF to HF power (LF/HF) of the instantaneous HR spectra were calculated for linear analysis. The largest Lyapunov exponent (LLE) of the R-R interval time series was calculated to determine a nonlinear property of HR. Infants at risk had significantly lower LF power (median: 0.51 x 10(-3) vs. 1.16 x 10(-3) Hz2) and lower LF/HF (median: 1.05 vs. 1.94) during quiet sleep. LLE was positive, revealing low-dimensional chaotic behavior of HR control, and did not differ between both groups (median: quiet sleep, 0.05 bit/s vs. 0.06 bit/s; active sleep, 0.16 bit/s vs. 0.15 bit/s). Sleep state-related changes in spectral parameters and LLE were similar in both groups. In infants at risk, the lower LF/HF during quiet sleep can be interpreted in terms of changes in the rhythmic components of the sympathovagal balance of the autonomic system, which is an expression of linear properties of HR control. Conversely, the lack of differences in LLE between both groups indicates similar nonlinear properties of the control system.

Does low frequency power of arterial blood pressure reflect sympathetic tone?

We tested whether power spectral analysis of arterial blood pressure (ABP) is a feasible tool to detect differences in peripheral sympathetic nerve activity in normotensive and hypertensive rats with differing basal sympathetic tones. Nine Wistar Kyoto rats (WKY), 10 Sprague-Dawley rats (SD), 10 spontaneously hypertensive rats (SHR) and 9 hypertensive transgenic rats harbouring the mouse Ren-2 gene (TGR) were chronically instrumented with femoral artery catheters and nerve electrodes around the sympathetic major splanchnic nerve. Two days after surgery ABP and splanchnic nerve activity (SpNA) were recorded in the conscious state during basal conditions as well as during alpha 1-adrenergic receptor blockade. Power spectra and squared coherence in the low (LF, 0.02-0.20 Hz), mid (MF, 0.20-0.80 Hz) and high (HF, respiration peak +/- 0.3 Hz) frequency bands were calculated for ABP and SpNA. Mean blood pressure in SHR (133 +/- 8 mmHg) and TGR (142 +/- 8 mmHg) was significantly higher (P < 0.05) than in WKY (115 +/- 3 mmHg) and SD (95 +/- 4 mmHg). SpNA in SHR was higher than in WKY (23.4 +/- 6.4 microV vs. 11.6 +/- 0.8 microV, P < 0.05) while SpNA in TGR was lower than in SD (20.1 +/- 3.9 microV vs. 28.8 +/- 4.2 microV, P < 0.05). LF and MF components of ABP variability were not significantly higher in those rats with high sympathetic tones. However, alpha 1-adrenergic receptor blockade reduced LF and MF components of ABP and SpNA in all strains except SHR. LF and MF coherence was not greater in rats with high sympathetic tones than in those with low sympathetic tones. The reduction of LF and MF components of ABP variability by alpha 1-adrenergic receptor blockade indicates an important contribution of peripheral sympathetic nerve activity to LF and MF blood pressure variability on an acute basis. However, the lack of higher LF and MF power in the ABP spectra of those rats with high SpNA together with the finding that LF and MF coherence was not higher in those rats with high SpNA led to the conclusion that LF and MF spectral components of ABP do not appear to be suitable markers for the prevailing sympathetic nerve activity.

Polarized expression of functional rat liver asialoglycoprotein receptor in transfected Madin-Darby canine kidney cells.

The rat liver asialoglycoprotein receptor or rat hepatic lectin (RHL) consists of two polypeptide species, a major one designated RHL-1 and a minor one designated RHL-2/3, which exists in two differentially glycosylated forms. We have studied the biosynthesis, targeting, and function of the different forms after transfection of their cDNAs into the polarized Madin-Darby canine kidney cell line. In cells expressing only RHL-1, newly synthesized protein undergoes rapid intracellular degradation and is not detected at the cell surface. In contrast, RHL-2/3 when transfected alone is much more stable and is expressed at the basolateral surface of fiber-grown cells. When both forms are expressed together, newly synthesized RHL-1 escapes rapid degradation and is detected at the basolateral surface. In double transfectants a functional receptor is formed that specifically endocytoses and degrades ligand at the basolateral side.