Freitag, 02.10.2020

14:00 - 15:30

Poster DGfN 2020

Hypertensiologie (P085 - P097)

Age attenuates the T-type CaV3.2-RyR axis in vascular smooth muscle

M. Kaßmann, G. Fan, Y. Cui, C. Matthaeus, S. V. Kunz, C. Zhong, D. Tsvetkov, O. Daumke, Y. Huang, M. Gollasch; Berlin, Hong Kong/CN, Greifswald

Objective: Caveolae position CaV3.2 (T-type Ca2+ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large-conductance Ca2+-activated K+ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca2+ spark generation is affected by age.
Method: Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both CaV3.2 channel inhibition by Ni2+ (50 μM) and caveolae disruption by methyl-ß-cyclodextrin or genetic abolition of Eps15 homology domain-containing protein (EHD2) inhibited Ca2+ sparks in cells from young (4 months) but not old (12 months) mice.
Results: Expression of CaV3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO CaV1.2−/− mice, caffeine (RyR activator) and thapsigargin (Ca2+ transport ATPase inhibitor), we found that sufficient SR Ca2+ load is a prerequisite for the CaV3.2-RyR axis to generate Ca2+ sparks. We identified a fraction of Ca2+ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd3+ (100 μM), but insensitive to CaV1.2 and CaV3.2 channel blockade.
Conclusion: Our data demonstrate that the VSMC CaV3.2-RyR axis is down-regulated by aging. This defective CaV3.2-RyR coupling is counterbalanced by a Gd3+ sensitive Ca2+ pathway providing compensatory Ca2+ influx for triggering Ca2+ sparks in aged VSMCs.

Microbial Colonization Protects From Hypertensive Cardiorenal Damage

H. Bartolomaeus, E. Avery, T. U. P. Bartolomaeus, U. Löber, D. Tsvetkov, K. Kräker, S. Y. Geisberger, L. Marko, B. Andràs, K.-U. Eckardt, S. Forslund, D. N. Müller, N. Wilck; Berlin

Objective: The gut microbiome is suspected to play a role in hypertension and hypertensive target organ damage. In the present study, we used germ-free mice to demonstrate that colonization with microbiota modulates the pathogenic response to a hypertensive stimulus.
Method: Four-week-old male germ-free C57BL6/J littermates were randomized either to remain germ-free (GF) or to receive bacterial colonization with regular specific pathogen free (SPF) microbiota from C57BL6/J mice (COL). At 12 weeks, hypertension was induced by implantation of an Angiotensin (Ang)II-liberating osmotic minipump (1.44mg/kg/d) and 1%NaCl in the drinking water; sham treated mice served as normotensive control. Two weeks after hypertension induction, we assessed inflammation and organ damage.
Results: Fecal and cecal bacterial load in COL mice was similar to conventionally raised SPF mice (16S rDNA qPCR). Shotgun metagenomic sequencing of fecal and cecal DNA revealed profound changes in response to AngII + 1%NaCl. Serum metabolomics using Biocrates MxP Quant 500 Kit were differentially altered by AngII + 1%NaCl in GF and COL mice. Hypertensive kidney damage was evident in both GF and COL mice. However, genes indicative of tubular damage (KIM-1, NGAL), inflammation (TNF, MCP-1), and fibrosis (Col3a2, Col1a3) showed a stronger increase in GF mice. Albuminuria and Masson’s trichrome staining confirmed the aggravated hypertensive kidney damage in GF mice. Similarly, we observed aggravated cardiac damage in GF mice. AngII + 1%NaCl induced greater cardiac hypertrophy (heart weight/tibia length) in GF compared to COL mice. BNP expression and echocardiography confirmed this finding. Histology of cardiac tissue showed aggravated fibrosis in GF mice. Only hypertensive GF mice showed a significant increase in inflammation-related gene expression (MCP-1, TNF). Immunofluorescence of cardiac tissue revealed a stronger increase of CD8+ cells in GF mice. Flow cytometry of splenic lymphocytes showed that anti-inflammatory myeloid-derived suppressor cells were depleted in sham as well as hypertensive GF mice. The adaptive immune response to AngII + 1%NaCl as evidenced by Th17 and CD8+ central memory T cells was exacerbated in GF mice. In vitro, naïve T cells isolated from GF mice more readily polarized towards Th17 cells compared to T cells from SPF mice.
Conclusion: Colonization status of mice profoundly affects the inflammatory response to hypertension. The magnitude of the pathogenic response to AngII + 1%NaCl in GF compared to COL mice demonstrates the importance of the gut microbiota in hypertensive target organ damage.

Changes In Gut Microbial Short-Chain Fatty Acid Production Potential Correlate With Blood Pressure Changes Under A Synbiotic Intervention

H. Bartolomaeus, T. U. P. Bartolomaeus, D. N. Müller, N. Wilck, S. Forslund; Berlin

Objective: Short-chain fatty acids (SCFA) are a class of metabolites produced by bacterial fermentation from otherwise indigestible dietary fiber. SCFA and high-fiber diet were shown to influence blood pressure levels and hypertensive target organ damage in experimental disease models. Here, we re-analyzed data from a published randomized, placebo-controlled trial investigating the effect of a synbiotic intervention in n=84 human subjects with metabolic syndrome (n=48) and healthy controls (n=26) (ISRCTN registry ID: ISRCTN37346212). For 3 months, subjects received a synbiotic containing live bacteria 108 CFU/daily dose and prebiotic fiber (2% inulin, 0.2% pectin) or a placebo. We hypothesized that changes in cardiovascular risk markers correlate with changes in SCFA synthesis capacity of the gut microbiome.
Method: Published metagenomic sequences were mapped to the KEGG database and then binned to gut metabolic modules (GMMs) using the GOMixer tool to estimate the gut microbial SCFA production potential for acetate, propionate, and butyrate. Data on blood pressure measurement and serum high-sensitivity C-reactive protein (hsCRP) before and after the intervention was similarly accessible. For in vitro validation of SCFA production, a synbiotic starter culture was grown on MRS agar in presence or absence of 2% inulin.
Results: On average, the synbiotic intervention compared to placebo did not significantly change hsCRP or blood pressure. SCFA production potential was similarly not influenced by the intervention. However, changes in diastolic blood pressure negatively correlated with changes in production potential of all three major SCFA in patients receiving the synbiotic. Similar trends were observed for systolic blood pressure and hsCRP. The synbiotic used in this study contained S. thermophilus, L. lactis, L. plantarum, L. fermentum, L. acidophilus, B. longum, and B. bifidum. Interestingly, after cultivation for 48hrs in presence of inulin, the consortium of bacteria produced measurable amounts of SCFA.
Conclusion: While more data from larger cohorts are needed, these data suggest that enhancement of intestinal bacterial SCFA production may ameliorate cardiovascular risk markers. Further clinical studies are needed to investigate the connection of dietary fiber, bacteria, and SCFA with blood pressure. Furthermore, future clinical studies focusing on microbiota should consider direct measurement of SCFA.

The differential impact of aerobic and isometric handgrip exercise on blood pressure variability and central aortic blood pressure

M. Seidel, N. Pagonas, F. Seibert, F. Bauer, B. Rohn, S. Vlatsas, D. Mühlberger, N. Babel, T. H. Westhoff; Herne, Berlin, Bochum

Objective: Blood pressure variability and central aortic blood pressure are independent markers of cardiovascular risk. Data on lifestyle-interventions to reduce these parameters are sparse. The present work reports the differential effects of aerobic vs. isometric handgrip exercise on blood pressure variability and central aortic blood pressure in a prospective randomized trial.
Method: 75 hypertensive patients were randomized to one of the following 12-week programs: A) Isometric handgrip training 5 times weekly; B) “Sham-handgrip training” 5 times weekly; C) Aerobic exercise training (30 minutes 3-5 times/week). Blood pressure variability was assessed by the coefficient of variation in 24h-ambulatory blood pressure monitoring. Central aortic blood pressure was measured non-invasively by the SphygmoCor device (AtCor Medical, Australia)
Results: The aerobic exercise program significantly decreased systolic daytime variability (12.1±2.5 vs. 10.3±2.8, p=0.04), whereas diastolic daytime blood pressure variability was not significantly altered (p=0.14). Nighttime variability was not significantly affected (p>0.05). Systolic central aortic blood pressure was reduced from 145.4±15.3 to 134.5±19.17 mmHg (p=0.01), diastolic central aortic blood pressure from 74.6±11.3 to 71.0±13.3 mmHg (p=0.2). Isometric handgrip and sham-handgrip exercise did not significantly affect blood pressure variability (p>0.05 each). Isometric exercise tended to reduce central aortic systolic blood pressure (142.1±19.44 to 135.8±17.44 mmHg, p=0.06). ANOVA revealed significant intergroup differences for the change of daytime systolic and diastolic blood pressure variability (p 0.048 and 0.047, respectively).
Conclusion: In contrast to aerobic exercise, isometric handgrip exercise does not reduce blood pressure variability but tends to lower systolic central aortic blood pressure in this hypertensive population.

The impact of calibration approaches on the accuracy of oscillometric central aortic blood pressure measurement

M. Glotzmann, M. Hogeweg, F. Bauer, F. Seibert, B. Rohn, M. Seidel, A. Mügge, N. Babel, T. H. Westhoff; Bochum, Herne

Objective: Two fully automated oscillometric devices have become available for the non-invasive assessment of central aortic blood pressure (BP). They tend, however, to underestimate systolic BP. It has been proposed that calibration by mean/diastolic instead of systolic/diastolic brachial BP may reduce this bias. The present work compares the accuracy of these two calibrations in the Mobil-O-Graph.
Method: Post-hoc analysis of the largest validation study on non-invasive assessment of central BP so far. Data on both calibration approaches were available in 159 patients without atrial fibrillation, who underwent simultaneous invasive and non-invasive assessment of central BP. Non-invasive BP measurements were conducted using the SphygmoCor XCEL (calibration by s/d brachial BP only) and the Mobil-O-Graph (calibration by both s/d and m/d brachial BP).
Results: Measurements of both devices and both calibration methods revealed highly significant correlations for systolic and diastolic central BP with invasively assessed BP (p<0.001 each). Calibration by m/d and s/d BP yielded similar correlations for central diastolic BP (R2 0.56 vs R2 0.55, p=0.919). Correlation of central systolic BP, however, was significantly lower using calibration by m/d brachial BP (R2 0.86 vs R2 0.74, p=0.002). Numerically, the SphygmoCor device revealed the highest correlation (R2 0.92 for central systolic and 0.72 for central diastolic BP; p<0.001 each). Calibration by s/d brachial BP was associated with an underestimation of central systolic BP using both the SphygmoCor and the Mobil-O-Graph. Calibration by m/d brachial BP, instead, was associated with an overestimation, which was numerically comparable (4.8±11.3 vs. -4.2±8). The calibration method had little effects on the biases of diastolic measurements.
Conclusion: Calibration by m/d instead of s/d brachial BP led to an overestimation instead of underestimation of central systolic BP without improving accuracy. Hence, m/d calibration is not necessarily superior to s/d calibration and the optimal approach has to be determined in a device-specific manner.

Impact of weather changes on hospital admissions for hypertension

F. Bauer, J. Lindtke, F. Seibert, B. Rohn, M. Seidel, N. Babel, P. Schlattmann, T. H. Westhoff; Herne, Jena

Objective: Blood pressure (BP) shows a seasonal variation with higher levels at lower temperatures. Many hypertensives, however, report on BP disturbances rather in association with acutely changing weather conditions than with absolute temperatures. To date, the impact of changing meteorological parameters on hypertensive episodes remains elusive.
Method: We performed a retrospective analysis on 203.703 patients in three hospitals in Germany, between 2010 and 2018, of whom 7.362 patients were admitted for hypertensive disease. Numbers of daily admissions for hypertension were associated with metereological data obtained from three near-by weather stations. Data comprised temperature (mean, maximal, minimal and range within 24h), athmospheric pressure, and rainfall. Changes of these parameters were calculated over a two and three day period.
Results: There was a significant inverse correlation between mean, maximal, minimal and 24h-range of temperature and the number of admissions for hypertensive disease. Moreover, rainfall was positively related to the number of admissions, whereas athmospheric pressure was not. With regard to all observed metereological parameters, neither the change within two, nor within three days was associated with the number of daily submissions for hypertensive disease.
Conclusion: Low temperatures are associated with higher numbers of hypertensive episodes requiring hospital admission. In contrast to the subjective perception of many hypertensive patients, however, changing weather conditions are not associated with a higher risk of hypertensive emergency.

Role of human T cells in resistant hypertension

M. Rahman, L. Hering, M. Yakoub, P. Heilmann, C. Schmidt, B. Duvnjak, J. Fischer, O. Vonend, L. C. Rump, J. Stegbauer; Düsseldorf, Berlin, Wiesbaden

Objective: Over-activation of the sympathetic nervous system (SNS) is a major contributor for resistant hypertension. Beside its direct effects on vasoconstriction and renal sodium handling, the SNS has significant influence on inflammation and immune cell response. In addition, animal studies support the concept that T cells play a role in the development of hypertension and hypertensive endorgan damage.
Method: Here, we analyzed differences in inflammatory markers and T cell subsets between patients with resistant hypertension (rHTN) and normotensive controls. Moreover, by using an immunodeficient mouse model, we examine the role of human T cells in the development of hypertension. To test this, we transfered T cells from patients with rHTN and healthy controls into immunodeficient NOD.Cg-Prkdcscid H2-K1tm1Bpe H2-D1tm1Bpe Il2rgtm1Wjl/SzJ (NSG-(KbDb)null) mice and induced experimental hypertension by chronic angiotensin (Ang)II (500ng/kg/min) infusion for 14 days. Blood pressure was continuously measured by radiotelemetry.
Results: In patients with rHTN, hsCRP, IL-6 and TNF-α as well as proportions of effector memory CD4 (CD4+CD45RA-CD62L-CCR7-), effector CD4 (CD4+CD45RA+CD62L-CCR7-) and CD8 (CD8+CD45RA+CD62L-CCR7-) T cells are significantly increased compared to healthy controls. To investigate whether these differences in T cell signature affect the development of hypertension, peripheral blood mononuclear cells (PBMC)s from rHTN or healthy controls were transferred to NSG-(KbDb) null mice. Systolic blood pressure did not differ at baseline between both groups (128± 5 vs. 135± 4mmHg, n=6). However, systolic blood pressure in response to AngII was increased in NSG-(KbDb)null mice receiving PBMCs from rHTN compared to healthy controls (week 1: 139±6 vs. 162±3mmHg, p<0.01; week 2: 140±6 vs. 159±7mmHg, p=0.08, n=5-6). In the kidney, perivascular T cell infiltration as well as mRNA expression of cytokines derived from human T cells such as TNF-α (1.5±0.2 vs. 1.0±0.1, p<0.05, n=5-6), IFN-γ (1.7±0.4 vs. 1.0±0.1, p=0.07, n=8-5), IL-17a (3.7±1.7 vs. 1.0±0.2, p=0.07, n=4-6) was relatively higher whereas TGF-ß expression (0.7±0.1 vs. 1.0±0.1, p<0.05, n=5-8) was significantly lower in NSG-(KbDb) null mice engrafted with PBMCs from rHTN compared to controls.
Conclusion: The present results suggest that T cells from patients with rHTN may directly influence the development of hypertension and therefore may have a pathophysiological relevance in the genesis of human hypertension.

The impact of treatment of period limb movements in sleep on blood pressure

D. Toma, M. Seidel, A. Doevelaar, F. Bauer, F. Seibert, B. Rohn, N. Babel, N. Büchner, S. Wang, T. H. Westhoff; Herne, Duisburg

Objective: Improving sleep quality in patients with obstructive sleep apnea by positive airway pressure therapy is associated with a decrease of blood pressure (BP). It remains elusive, however, whether treatment of sleep disturbances due to period limb movements in sleep (PLMS) affects BP as well. The present study provides first data on this issue.
Method: Retrospective study on 114 patients undergoing in-laboratory polysomnography in a German University Hospital. Inclusion criteria were first diagnosis of PLMS with a periodic leg movement index ≥ 15/h and periodic leg movement arousal index ≥ 5/h with subsequent initiation of therapy with dopamine or dopamine agonists. Exclusion criterion was an initiation or change of preexisting positive airway pressure therapy between baseline and follow-up polysomnography. BP and Epworth sleepiness scale (ESS) were assessed at two consecutive in-laboratory polysomnographies. BP was assessed auscultatorily according to Riva-Rocci at admission
Results: Mean age of the study population was 62.1±12.1 years. 90 patients (78.9%) suffered from hypertension, 89 (78.0%) had antihypertensive medication. 76 subjects (66.6%) had preestablished CPAP therapy. All the patients were diagnosed to have PLMS. 100 patients (87.7%) were started on dopamine (mean dosage 159.0±67.8 mg), 14 patients (12.2%) on dopamine agonists after the baseline examination (pramipexole, mean dosage 0.3±0.1 mg, ropinirol 2.5±1.5 mg). ESS significantly decreased from baseline to follow-up from 6 (interquartile range (IQR) 3 – 10.5) to 5 (IQR 3 - 10, p 0.013). Systolic BP significantly decreased from 132.9±17.1 to 127.1±19.2 mmHg (p 0.004), whereas there was no change of diastolic BP (75.9±12.5 vs. 75.1±9.2 mmHg, p 0.48). The number of antihypertensive drugs remained unchanged with a median of 2 at baseline and follow-up (p 0.27).
Conclusion: Treatment of PLMS by dopamine or dopamine agonists is associated with an improvement of sleep quality and a decrease of systolic but not diastolic BP.

Calcium Signals in Murine Glomerulosa Cells of Cacna1h Knock-out Mice

H. A. Dinh, U. Scholl, G. Stölting; Berlin

Objective: The steroid hormone-producing cortex of the adrenal glands consists of the inner zona reticularis, zona fasciculata and the outer zona glomerulosa (ZG), producing androgens, cortisol and aldosterone, respectively. Aldosterone plays a crucial role for the maintenance of serum electrolytes as well as blood pressure by increasing the reabsorption of NaCl along with water in the kidney. The two main physiological stimuli of aldosterone synthesis are angiotensin II (AT-II) and hyperkalemia. Stimulation of ZG cells leads to oscillatory membrane depolarizations. These depolarizations are assumed to be linked to the influx of calcium through the activation of voltage-gated calcium channels in addition to the release of calcium from activated IP3-sensitive calcium stores. This results in an increase of the intracellular calcium concentration ([Ca2+]i), which provides the signal for aldosterone production. It has been reported that voltage oscillations require CaV3.2 (Gene: Cacna1h) but it is unclear what the importance of this channel for the influx of calcium is.
Method: Loading of acute slice preparations of adrenal glands from Cacna1h knockout (KO) and wild-type mice with the ratiometric indicator dye Fura-2 AM allowed for absolute measurements of [Ca2+]i. Effects on oscillations were confirmed using the intensiometric dye Calbryte 520 AM, which allows for better temporal resolution. We studied the response of these parameters to stimulation with AT-II and K+ as well as the dependence of oscillations on extracellular Ca2+.
Results: ZG cells from WT and KO mice exhibited fast oscillatory changes of [Ca2+]i clustered in bursts. On average, [Ca2+]i was up to 21% lower during activity and at baseline in cells from KO compared to WT mice. Within bursts, the spiking frequency was higher in cells from KO than WT mice (1.38 ± 0.98 Hz in KO mice versus 0.47 ± 0.16 Hz in WT mice, mean ± SD). Recordings from WT and KO mice support a positive correlation between the extracellular [K+] and [AT-II] and [Ca2+]i in both. Removal of extracellular Ca2+ led to the absence of signals in both WT and KO mice in less than 10 minutes, and baseline [Ca2+]i values were lowered.
Conclusion: Mean [Ca2+]i as well as baseline and burst [Ca2+]i values were lower in KO than WT mice, suggesting that CaV3.2 contributes to overall [Ca2+]i. However, cells from KO mice still exhibited oscillatory activity, indicating that additional calcium channels play a role in ZG calcium signal generation. To elucidate the source of the remaining calcium signals in the absence of CaV3.2, further work is required.

SP - differential influences on action potential production in afferent neurons of the kidney?

K. Rodionova, T. Ditting, K. F. Hilgers, C. Ott, R. E. Schmieder, M. Schiffer, K. Amann, R. Veelken; Erlangen

Objective: Afferent nerve fibers of the kidney play a role in controlling sympathetic activity in hypertension and cardiovascular diseases. Proinflammatory substances influence the action potential production of these neurons. Therefore, we tested the hypothesis that proinflammatory substance P (SP) released from afferent nerves inhibits the action potential production in neurons with renal afferents.
Method: Cultured dorsal root ganglion neurons (DRG Th11-L2) of rats with renal afferents were investigated in current clamp mode to assess action potential generation during both current injections and TRRPV1 stimulation with protons (pH 6) with and without exposure to SP (0.5 µmol) or CGRP (0.5 µmol). Neuronal classification as tonic (high AP generation upon stimulation) and phasic (AP ≤ 5 upon stimulation). Additional experiments were performed in voltage clamp mode to fully assess electrophysiological properties of the neurons.
Results: : Renal neurons were stimulated with current injection (14.4+/-1.5 APs/600ms, mean+/- SEM) and protons (9,6+/-1,9 APs/10s of stimulation with pH6). The co-stimulation of renal neurons with current injections and SP decreased the number of action potentials in tonic neurons (15,2+/-1.1 APs/600ms vs. 10.1+/-1.6 APs/600ms, p<0.05, mean+/- SEM), however superfusion of renal neurons with both protons (pH 6) and SP increased it (9.6+/-1.9 APs/10s vs. 16.9+/-2.3 APs/10s, p<0.05, mean+/- SEM). Addition of SP itself did not stimulate cultivated neurons. Co-stimulation with CGRP (second proinflammatory transmitter contained in afferent renal nerve fibers) was without significant effect under any circumstances.
Conclusion: Neuronal SP influences action potential production in renal neurons in a very complex way: Both inhibition and specific increases in action potentials via a TRPV1-dependent mechanism in acid-sensitive renal neurons could be demonstrated. Afferent nerve fibers are likely to respond very specific in different conditions while influencing sympathetic nerve activity and putatively renal physiology or pathology (proinflammatory actions of SP).

Malignant hypertension in renovascular hypertensive rats: possible therapeutic role for hypoxia-inducible factor stabilization ?

K. F. Hilgers, T. Dambietz, N. Cordasic, G. Schley, C. Willam, N. Burzlaff, M. Schiffer, K. Amann, R. Veelken, A. Hartner; Erlangen, Nürnberg

Objective: We previously reported impaired angiogenesis in the malignant versus non-malignant course of renovascular hypertension in rats. We now tested the hypothesis that stimulating angiogenesis by stabilization of hypoxia-inducible factor (HIF) would prevent the development of malignant hypertension and target organ damage.
Method: Two-kidney, one-clip renovascular hypertension (2K1C) was induced in 33 rats; controls (CON) were sham operated. 14 2K1C received intraperitoneal injections of 12.5 mg/kg of the prolyl hydroxylase inhibitor ICA on days 15, 16, 18 and 19 to stabilize HIF; 19 2K1C received only vehicle. Rats were sacrificed on day 35. To distinguish malignant from non-malignant hypertension, we considered two factors: weight loss, and the occurrence of typical vascular lesions (onion skin lesions and fibrinoid necroses) in the nonclipped kidney. We assessed capillary density (after RECA staining) as well as kidney damage in the nonclipped kidney. In a separate experiment, 10 2K1C receiving ICA and 10 hypertensive rats receiving vehicle were sacrificed on day 19 to assess HIF staining in target organs.
Results: Mean blood pressure measured intraarterially in conscious rats was elevated to the same degree in ICA-treated (212±8 mmHg) and vehicle-treated (221±6 mmHg) 2K1C compared to CON (128±9 mmHg, p<0.01). In the ICA group, 6 of 14 2K1C rats developed malignant hypertension, as did 9 of 19 vehicle-treated 2K1C (p=0.74 by χ2). Malignant 2K1C displayed more severe heart hypertrophy and kidney damage but there was no effect of ICA treatment. The number of capillary cross sections per high-power view tended to be reduced in all 2K1C (p=0.06) compared to controls but there was no difference between ICA-treated and vehicle-treated 2K1C, respectively. ICA increased the number of HIF-positive nuclei in renal medullary tissue (72±10 versus 27±14 nuclei per high power field in vehicle treated 2K1C, p<0.017).
Conclusion: Intermittent HIF stabilization did not affect blood pressure, capillary rarefaction, hypertensive target organ damage, or the development of malignant hypertension in renovascular hypertension in rats.

Effect of sunitinib treatment on skin sodium accumulation in patients with renal cancer: a pilot study

L. Marko, A. Dörr, P. Linz, A. H. van den Meiracker, T. Kühne, R. Dechend, A. H. J. Danser, A. Flörcken, D. N. Müller; Berlin, Erlangen, Rotterdam/NL

Objective: Angiogenesis is required for invasive tumor growth and metastasis which is controlled mainly by vascular endothelial growth factors (VEGF). Novel strategies for cancer treatment target VEGF signaling. These agents are featured by adverse events including hypertension. New concepts suggest that besides the kidneys, the skin also plays a role in body sodium homeostasis and blood pressure regulation by a VEGF-C–dependent buffering mechanism. Here, we tested the hypothesis that changes in blood pressure correspond to tissue sodium accumulation during sunitinib treatment of metastatic renal cell carcinoma patients ( NCT04368546; Charité ethical approval EA1/044/15).
Method: Male patients (n=4) took sunitinib according to the standard treatment protocol, 50 mg once daily, taken for 4 weeks followed by a 2-weeks off treatment period. Measurements were performed at baseline (before sunitinib treatment), at the end of the first on-treatment phase, at the end of the first off-phase and finally at the end of the second on-treatment phase. Tissue sodium content was measured using non-invasive 23Na magnetic resonance-imaging (MRI) technology; skin sodium was measured in a group (n=5) of age-matched healthy subjects, as well. Blood pressure was measured after 15 minutes of sitting for 20 minutes at 5 minutes intervals using an automatic ambulatory blood pressure monitor and building average of the last 3 measured values. Blood withdrawal followed after ca. 45 minutes of sitting to measure VEGF-A, VEGF-C, endothelin-1, renin and aldosterone levels.
Results: Elevated systolic blood pressure under sunitinib treatment decreased to the baseline level in the off-treatment phase (130.5 mmHg vs 117.5 mmHg, respectively, p<0.05). Plasma endothelin-1 levels mirrored directly the blood pressure changes whereas VEGF-A levels inversely. Renin and aldosterone levels did not show any trend. Baseline skin sodium content was similar to the skin sodium level of healthy controls. Skin sodium content was elevated after the first on-treatment phase and stayed high in all the following measurements in comparison to control subjects. Patients had high baseline VEGF-C levels which decreased after the first treatment with sunitinib and stayed low independently of further treatment phase.
Conclusion: In conclusion, sunitinib treatment suppresses VEGF-C levels permanently which is associated with sustained elevated skin sodium levels. Furthermore, we confirmed the association between plasma endothelin-1 levels and blood pressure changes during sunitinib treatment protocol.

The Role of Clcn2 in the Murine Zona Glomerulosa

M. Volkert, U. Scholl, G. Stölting; Berlin

Objective: The zona glomerulosa (ZG) comprises the outer layer of the adrenal cortex, which synthesizes aldosterone. This steroid hormone regulates the volume and electrolyte balance of the body as well as the blood pressure. The serum concentrations of potassium and angiotensin II (AT-II) are the most important stimuli for aldosterone synthesis in the ZG. They lead to oscillatory depolarizations and cause calcium influx into ZG cells, which is the signal for aldosterone production. Aldosterone synthesis can also be increased by other means like changes in serum osmolality. Among mutations in other genes, heterozygous gain-of-function mutations in CLCN2, which encodes the chloride ion channel ClC-2, lead to familial hyperaldosteronism. Mouse models with such mutations show stronger depolarization and increased calcium signaling of the ZG. The role of ClC-2 in the healthy ZG, however, is still unclear. While it is possible that ClC-2 is directly involved in the electrical origins of calcium influx, many of the identified mutations are located in regions that have been described in connection with the osmosensitivity of ClC-2.
Method: In order to determine the role of ClC-2 in the ZG, Clcn2 knockout (KO) and wild type mice were compared. Some results were validated in mice with a mutation linked to primary hyperaldosteronism, ClC-2 R180Q. In acute slice preparations of murine adrenal glands, the cytosolic calcium concentration was examined with intensio- or ratiometric fluorescent calcium indicators. We studied the effects of different extracellular potassium and AT-II concentrations as well as various osmotic conditions regarding on oscillations in intracellular calcium concentrations.
Results: We confirmed that calcium oscillations in ZG increased with higher extracellular potassium and AT-II concentrations. After an acute change to a hypoosmolar solution, the wild type initially showed a higher activity than the KO mouse. Following a prolonged exposure to an extracellular hypoosmolar condition, however, the activity in the ZG of the KO mouse was more than 1.4 times higher compared to the wild type. A rapid increase in extracellular osmolality led to an acute decrease in activity by up to 35 % for the wild type and 72 % for the KO.
Conclusion: Our results suggest that ClC-2 plays a role in the response of the ZG to changes in extracellular osmolality. Further work is still needed to clarify how the observed effects are mediated at the molecular level in KO and wild type mice.


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