Angiotensin II (Ang II), a peptide hormone involved in the development of hypertension, causes systemic and cerebral inflammation, affecting brain regions important for blood pressure control. The cause-and-effect relationship between hypertension and inflammation is two-way, but the role of blood pressure in the induction of cerebral inflammation is less clear. The vulnerability of specific brain regions, particularly those important for memory, is also of interest.We used molecular biology approaches, immunohistochemistry, and electron microscopy to examine the interdependence between the hypertensive and pro-inflammatory effects of Ang II. We examined the effect of blood pressure by administering a subpressive (200 ng/kg/min) or a pressive Ang II dose (1000 or 1900 ng/kg/min) with and without hydralazine (150 mg/L) for 1 week and used phenylephrine to increase blood pressure independently of the renin-angiotensin system.Ang II increased ionized calcium-binding adaptor molecule 1 (Iba-1) levels (marker of microgliosis) in the whole brain and in the hippocampus in a dose-dependent manner. Pressive Ang II induced specific changes in microglial morphology, indicating differences in functional phenotype. An increase in hippocampal glial fibrillary acidic protein (GFAP) was seen in mice receiving pressive Ang II, while no induction of cerebral gliosis was observed after 7 days of subpressive Ang II infusion. Although phenylephrine led to increased astrogliosis, it did not affect Iba-1 expression. Pressive Ang II stimulated TNF-α production in the hippocampus, and daily treatment with hydralazine prevented this increase. Hydralazine also reduced GFAP and Iba-1 levels. With longer perfusion (14 days), subpressive Ang II led to some but not all the inflammatory changes detected with the pressive doses, mainly an increase in CD68 and Iba-1 but not of GFAP or TNF-α.Blood pressure and Ang II differentially contribute to hippocampal inflammation in mice. Control of blood pressure and Ang II levels should prevent or reduce brain inflammation and therefore brain dysfunctions associated with hypertension.The online version of this article (10.1186/s12974-018-1090-z) contains supplementary material, which is available to authorized users. Hypertension is a serious public health issue, being highly prevalent in the elderly  and a leBesides reflecting an increase in arterial blood pressure, hypertension consists of a chronic, low-grade inflammation involving innate and adaptive immune cells . The linStudies examining the role of cerebral inflammation in experimental hypertension have focused on the paraventricular nucleus (PVN), a region of the hypothalamus involved in the sympathetic control of blood pressure . Using aGiven the strong associations between mid-life hypertension and late-life dementia –21, the Therefore, this study was set to answer these questions. We administered Ang II in the systemic circulation of C57BL/6 mice during 7 or 14 days and examined the inflammatory response in the whole brain and in the hippocampus using a multidisciplinary approach. In order to dissect the effects of Ang II and blood pressure, we delivered pressive versus subpressive Ang II doses and phenylephrine, or used the peripheral vasodilator hydralazine to prevent hypertension. Our results show that Ang II and blood pressure can differentially contribute to the development of hippocampal inflammation in mice.Ten to 12-week-old male C57BL/6 mice  were housed individually in a temperature-controlled room with a 12-h light-dark cycle and access to food and water ad libitum. Following acclimatization, mice were randomly assigned to experimental groups. The precise number of animals used for each experiment is specified in the Escherichia coli, 2 mg/kg, Sigma-Aldrich, Oakville, Canada) and sacrificed 3 h later.Alzet osmotic minipumps  were subcutaneously implanted as previously described . Mice reBlood pressure was monitored by non-invasive tail-cuff plethysmography . Blood pressure assessment began 3 days before minipump implantation in order to habituate the mice to the procedure and continued at three different intervals  during 1 week. Mice were placed on a heating platform for 10 min before assessment of blood pressure. A minimum of five measurements were taken until the blood pressure stabilized and, following stabilization, a minimum of eight measurements were taken per mouse and averaged for analysis. Blood pressure was monitored by the same person at the same time of the day.Equal amounts of proteins from whole brain homogenates (75 μg) were subjected to electrophoresis and transferred to nitrocellulose membranes . Membranes were probed with rabbit anti-Iba-1  or mouse anti-GFAP  antibodies overnight at 4 °C. Rabbit anti-pan actin  was used as loading control. Densitometric analysis was performed using ImageJ software , and the ratio Iba-1/actin or GFAP/actin was expressed relative to the control group. Details on tissue preparation and additional details on the Western blot protocol are available in Additional file Details on tissue preparation are available in Additional file Details on tissue preparation are available in Additional file Immunostaining was performed on sections containing the dorsal hippocampus (from Bregma − 1.46 to − 1.55 mm). Details on tissue processing and staining are available in the Additional file TNF-α and IL-6 were assessed by ELISA  in the brain or hippocampal homogenates as well as in plasma samples, as described in Additional file t test, as appropriate. Spearman rank correlation was used to examine the association between TNF-α and blood pressure. A value of p ≤ 0.05 was considered statistically significant. For each experiment, the statistical analysis is specified in the figure legends and tables. Data is presented as mean ± SEM.Data analysis was done with GraphPad Prism v.7.0 . Multiple group comparisons were evaluated by one-way or two-way analysis of variance (ANOVA), with the Tukey post hoc test. Two-group comparisons were analyzed with the Mann-Whitney test or a two-tailed Subcutaneous perfusion of Ang II at a concentration of 1000 ng/kg/min resulted in a steady significant increase in systolic blood pressure (SBP) of 33.8 mmHg (mean increase) at day 4 and 37.1 mmHg (mean increase) at day 7, compared to day 0 (Table p = 0.035) or to mice receiving Ang II 200 (p = 0.034) , indicating either an increase in the number of microglia or phenotypical changes of these cells. GFAP protein levels followed a similar trend in response to pressive Ang II . No significant changes in cerebral gliosis were detected in mice receiving subpressive doses of Ang II after 7 days perfusion . Instead, a significant increase in GFAP mRNA expression was evident in mice that received the hypertensive dose of Ang II (p = 0.031) but not the subpressive dose . In line with the mRNA results, quantitative immunofluorescence showed comparable hippocampal CD68 levels between mice receiving subpressive Ang II and their controls, although a trend revealing a mean 16% increase in CD68 was seen with the pressive dose . Iba-1 showed a similar response . We then examined the impact of Ang II on hippocampal astrogliosis. Immunofluorescence analysis revealed a significant increase in GFAP levels of around 50% in response to pressive Ang II (p = 0.001) but not to the subpressive dose  that increased blood pressure to the same level of the dose of 1000 ng/kg/min. The second one examined the effects of phenylephrine, a hypertensive drug which is Ang II-independent. The third prevented Ang II-induced hypertension by simultaneously treating mice with hydralazine.p < 0.0001, Fig. n = 4–5), suggesting a dose-dependent effect of Ang II on microgliosis. The higher dose of Ang II (1900 ng/kg/min) also increased GFAP levels by 60% following the 7-day chronic administration .Despite a similar increase of blood pressure as the dose of 1000 ng/kg/min Tables , infusiop < 0.0001, Fig. n = 4–5), indicating that increased blood pressure alone can contribute to hippocampal astrogliosis.No change in hippocampal Iba-1 expression was detected with perfusion of phenylephrine Fig. a, even tp = 0.055, Fig. n = 3–6). Likewise, hydralazine reduced the elevation in hippocampal GFAP in mice receiving Ang II 1000  to levels comparable to controls, supporting the idea that increased blood pressure is an important contributor to Ang II-induced astrogliosis.With a different strategy to explore the effects of blood pressure, we looked at Iba-1 and GFAP levels in the hippocampus of mice receiving the hypertensive dose of Ang II (1000 ng/kg/min) and treated with the peripheral vasodilator hydralazine Fig. . Treatmep = 0.065, Additional file n = 3) and no differences in GFAP . While no differences in hippocampal CD68 had been seen with infusion of subpressive Ang II for 1 week, increasing Ang II perfusion to 2 weeks led to a significant rise in CD68 mRNA  and to a similar increase in GFAP mRNA (mean increase 29%), although this difference did not reach statistical significance .To rule out that the absence of brain inflammatory changes in mice receiving the subpressive dose for 1 week was not due to insufficient perfusion time, we examined a separate group of mice that received chronic Ang II infusion (200 ng/kg/min) during 14 days. Western blot analysis in the whole brain revealed a trend for a mean 35% increase in Iba-1 levels (n = 3–4). There was a 19% mean increase in both process area . We studied ultrastructural changes in microglial processes  as well as changes in their accumulation of vacuoles and extracellular digestion. EM analysis showed significant differences in microglia morphology between control and hypertensive mice receiving Ang II 1000 ng/kg/min  and IL-6 . Mice that received Ang II 200 ng/kg/min did not present elevations in peripheral plasma cytokines, neither after 1 week nor after 2 weeks perfusion .We next examined whether systemic Ang II would lead to increased production of pro-inflammatory cytokines in the brain. First, we confirmed that Ang II infusion increased the levels of the pro-inflammatory mediators TNF-α and IL-6 in plasma. As shown in Fig. n = 6–8). This was not due to a lack of detection capacity of our method as mice receiving a single systemic LPS injection showed significant increases in the production of TNF-α  and of IL-6  compared to controls . Even with longer perfusion times, no changes in TNF-α production in the whole brain were detected with infusion of Ang II 200 ng/kg/min for 2 weeks .Despite the robust systemic pro-inflammatory effect seen with hypertensive Ang II, ELISA analysis did not detect an elevation of these cytokines in the whole brain with any of the doses Fig. n = –8. This p = 0.040, Fig. n = 6–9). Supporting the effects of blood pressure on cerebral inflammation, hydralazine treatment prevented the increase in hippocampal TNF-α production triggered by hypertensive Ang II .We next considered whether the apparent absence of increased cytokine production in the brain of mice chronically infused with Ang II was due to the presence of a specific regional change, which was diluted by analysis of the whole brain. We decided to examine TNF-α in more detail rather than IL-6, based on previous evidence that hypothalamic microglia isolated from Ang II-treated mice exhibit a significant increase in TNF-α expression and a milder increase in IL-6 , 18. In r 0.658, p = 0.017), Spearman analysis did not reveal a significant correlation between these two parameters when TNF-α was assessed in the brain (r 0.456 p = 0.102), suggesting a non-linear relationship between blood pressure and cerebral inflammation.We further examined the association between hippocampal TNF-α and systolic blood pressure via correlation analysis. While a significant positive association was found between the concentration of TNF-α measured in plasma and blood pressure . With these approaches, our results show that circulating Ang II leads to hippocampal inflammation and cerebral gliosis in a dose-dependent manner and that increased blood pressure is an important contributor to the brain pro-inflammatory effects of Ang II in mice.Is blood pressure elevation alone sufficient? With administration of phenylephrine, we did not observe an increase in Iba-1, as we have seen with the highest hypertensive dose of Ang II, even though the dose of phenylephrine was ten times higher than that of Ang II (19 μg/kg/min versus 1900 ng/kg/min). In addition, analysis showed a non-linear correlation between hippocampal TNF-α and blood pressure, suggesting that other factors could modulate the association between these parameters. For example, the breakdown of the blood-brain barrier (BBB) induced by Ang II may be involved in the inflammatory process , 32. IndDespite bypassing the renin-angiotensin system, we observed a significant increase in GFAP expression in the hippocampus with phenylephrine treatment. This vulnerability of astrocytes is not surprising considering their anatomical proximity to cerebral blood vessels as part of the neurovascular unit , being cBeyond our study, a critical role for Ang II on cerebral inflammation is supported by a previous work showing that inhibition of AT1 receptors with candesartan, a centrally acting angiotensin receptor blocker, leads to reductions in brain inflammation induced by LPS or stroke in normotensive rats , 37. At With respect to hydralazine, there have been reports indicating it exhibits antioxidant properties beyond its well-recognized antihypertensive effect, including scavenger actions on peroxynitrite and oxygen radicals –41. AlthPrevious studies examining the cerebral pro-inflammatory effects of pressive Ang II focused on the PVN, a region of the hypothalamus involved in the sympathetic control of blood pressure . These sBesides an upregulation of TNF-α and astrogliosis, we report significant morphological changes of microglia in the hippocampus of mice infused with pressive Ang II, as detected by immuno-EM. Changes in microglial morphology are informative of their functional activation state in response to stimuli or injury. In this study, we labeled microglia with Iba-1, which is a well-recognized marker specifically expressed by resident microglia and macrophages , 44. We Bdnf and Homer1 [The increase in process area and perimeter would indicate that they are larger in the presence of Ang II, reflecting process thickening and therefore increased metabolic or lysosomal activity. This is also supported by the finding of reduced solidity and thus of a more ruffled morphology, which could be a sign of increased process motility reflecting their capacity to reorient towards sites of injury . Furtherd Homer1 . We could Homer1 –21.It is interesting that such significant and specific changes in hippocampal microglia morphology were detected in the absence of increased expression of Iba-1 or CD68 in this region. These results highlight the sensitivity of immuno-EM ultrastructural studies to reveal alterations in microglia morphological dynamics that may otherwise appear “normal” when examined by protein or gene expression analysis. With respect to other regions, the increase in Iba-1 in the whole brain in response to pressive Ang II could be a signal of increased microglial numbers or phenotypic alterations taking place outside the hippocampus  or even in the white matter, an interesting hypothesis warranting future investigations.In addition to maintaining brain homeostasis, microglia undergo phenotypic transformation upon tissue injury, or in response to Ang II, leading to the secretion of inflammatory and oxidative molecules that contribute to neurodegeneration . A recenBesides affecting microglia, our finding of increased hippocampal GFAP expression in response to pressive Ang II indicates that astrocyte activation is also involved in Ang II-induced neuroinflammation. Indeed, in a very comprehensive study, Stern and colleagues recently showed that astrocytes are critical and direct cellular mediators of Ang II actions on PVN neurons, leading to increased sympathetic outflow and to increased blood pressure .Therefore, it is likely that the cerebral inflammation induced by Ang II involves multiple pathways and cellular players, including but not limited to (i) the direct effects of circulating Ang II on AT1 receptors in cerebral vessels ; (ii) thThe findings of our study must be considered within its limitations. One lesson learned from our work is that microglia morphology analysis via EM is able to detect subtler and even more informative changes than immunofluorescence, as seen by the lack of Iba-1 increase with the dose of 1000 ng/kg/min Ang II in parallel with significant changes in the morphology of microglia. In this regard, although Iba-1 levels in phenylephrine-treated animals were comparable to controls, we cannot discard the possibility that phenylephrine could have affected microglia morphology in this group. It would be interesting to examine whether metabolic changes in microglia differ upon Ang II or phenylephrine treatment despite similar effects on blood pressure. Also, our study examined only male mice and potential sex differences in the response to the pressive and inflammatory effects of Ang II cannot be discarded. Despite these considerations, this work broadens our understanding of the in vivo effects of Ang II actions in relation to hypertension and its impact on brain inflammation.Our results indicate that the cerebral pro-inflammatory effects of pressive Ang II involve changes in microglial morphology, as well as increases in cytokine production and GFAP expression in a region of the brain critical for memory. EM analysis revealed phenotypic alterations in microglia consistent with an active metabolic state, which could be involved in synaptic remodeling, in the absence of increases in Iba-1 or CD68 expression. This emphasizes the value of immuno-EM to reveal alterations in cerebral gliosis upon tissue injury. The findings of this study also indicate that increased blood pressure, in addition to Ang II itself, is an important contributor to the development of cerebral inflammation. Taken together, these results open the door for future investigations on the mechanisms underlying the induction of cerebral inflammation by hypertension, leading to new questions such as the following: What is the threshold of blood pressure necessary to induce cerebral inflammation? Which parameters influence this threshold? Can antihypertensive treatments reverse this inflammation? Answers to these questions may provide great advancements for a better understanding of the link between hypertension and cognitive decline as well as for the development of therapies against neurodegenerative diseases.Additional file 1:Supplementary Methods. (DOCX 59 kb)Additional file 2:Figure S1.. Effect of extended perfusion of subpressive Ang II on cerebral inflammation. Iba-1 (A) and GFAP (B) were examined by Western blotting in cerebral homogenates after 2 weeks systemic perfusion of Ang II 200 ng/kg/min or 0.9% saline (CTL). Densitometry values of Iba-1 and GFAP were normalized to actin, and the results are expressed relative to the control group. Representative immunoblots are shown . CD68 mRNA (C) and GFAP mRNA (D) gene expression were analyzed by qRT-PCR in microdissected hippocampi after 2 weeks systemic perfusion of Ang II 200 ng/kg/min or 0.9% saline (CTL). In each experiment, a treated-to-control ratio was calculated . ELISA analysis of TNF-α in plasma (E) and in whole brain homogenates (F) after 2 weeks perfusion of Ang II 200 ng/kg/min or 0.9% saline (CTL). A treated-to-control ratio was calculated . (TIFF 1442 kb)


Blood pressure is an extremely important blood hemodynamic parameter. The pulse wave contains abundant blood-pressure information, and the convenience and non-invasivity of its measurement make it ideal for non-invasive continuous monitoring of blood pressure. Based on combined photoplethysmography and electrocardiogram signals, this study aimed to extract the waveform information, introduce individual characteristics, and construct systolic and diastolic blood-pressure (SBP and DBP) estimation models using the back-propagation error (BP) neural network. During the model construction process, the mean impact value method was employed to investigate the impact of each feature on the model output and reduce feature redundancy. Moreover, the multiple population genetic algorithm was applied to optimize the BP neural network and determine the initial weights and threshold of the network. Finally, the models were integrated for further optimization to generate the final individualized continuous blood-pressure monitoring models. The results showed that the predicted values of the model in this study correlated significantly with the measured values of the electronic sphygmomanometer. The estimation errors of the model met the Association for the Advancement of Medical Instrumentation (AAMI) criteria  and the Grade A British Hypertension Society criteria. Blood pressure is the combined result of ventricular ejection and peripheral resistance. Blood pressure is divided into arterial blood pressure and venous blood pressure, and blood pressure without any specific descriptors generally refers to arterial blood pressure . HyperteAn accurate and real-time blood-pressure value is profoundly important for the diagnosis, prevention, and treatment of hypertension-related diseases . HoweverThe arterial tonometry method is applicable to superficial arteries such as the radial artery . CalibraIn recent years, scholars have extracted the features of the pulse wave signals in the time and frequency domains and constructed multivariate regression models ,20,21,22The present study extracted as many waveform characteristics related to blood pressure as possible based on PPG in combination with electrocardiography (ECG) signals, as well as an individual’s characteristic features. Furthermore, the neural network was used as the mainline, the impact of the extracted features on the model output was analyzed, and then an appropriate feature set was selected. Moreover, the blood-pressure estimation model was constructed based on the selected features and optimized with optimization algorithm. Finally, the estimation accuracies of the proposed models were explored according to the selected criteria and compared models.The experiments were approved by the Ethics Committee of the First Affiliated Hospital of Third Military Medical University, and the date of approval was 4 November 2013. and all subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki. The hardware platform for experimental data acquisition included a non-invasive cardiac function analyzer prototype developed independently in this study and an Omron electronic sphygmomanometer (HEM-7210) . The non-invasive cardiac function analyzer prototype was used to synchronously collect the fingertip photoplethysmogram signals and ECG signals of the individuals. A lead-II ECG configuration was used. The photoplethysmogram sensor was clamped onto the tip of the right index finger of the individual and kept at the same level with the heart, and the signal sampling frequency was set at 500 Hz. The Omron electronic sphygmomanometer was used to measure the blood pressure at the corresponding time. The cuff was worn on the left upper arm of the individual, and the electronic sphygmomanometer was used in strict accordance with the manuals .A total of 27 healthy subjects  aged 18–27 years were enrolled in this study. The recruitment of participants was based on voluntary and informed consent, and the participants had no conflicts of interest with research contents and results.The static and dynamic experiments were performed at room temperature, and the dynamic experiment was a random-number mental arithmetic . During The ECG and PPG signals are weak physiological signals susceptible to industrial frequency, tremor, and breathing during signal collection. The denoising of ECG signals and PPG signals is indispensable to ensure accurate extraction of signal characteristics. In this study, the wavelet packet method was employed to denoise the ECG and PPG signals on the MATLAB 2016b platform  , and theup) and to the f point of the dicrotic notch (tbf); the time from c and f to the point b of the next cardiac cycle ; the time from the point a with maximum ascending slope to the point e with minimum descending slope of the photoplethysmography (tae); the ratio of the aforementioned time parameters to the cardiac cycle ; as well as the time from the R-wave of ECG to the photoplethysmography points b, a, and c within the same cycle . The amplitude parameters were the ratio of the amplitude of the points a, e, f, and g to the amplitude of the point c . The area parameters were the systolic area Sbf and the diastolic area Sfb. Other types of parameters also existed, such as the K value (K = (Sm−Hc)/(Hc−Hb), Sm is the mean pulse wave area of the cycle), the heart rate (HR) calculated by the adjacent R-wave interval, and the main wave rising slope (Cslope). A total of 22 waveform characteristics were included, and a schematic diagram of some features is shown in The time parameters were extracted from the denoised ECG and PPG signals, including the time from the pulse starting point b to the peak point c (tThe error back-propagation (BP) algorithm has a simple network structure, a powerful parallel processing capability, and a strong fault tolerance, and can approximate the complex non-linear relationships well . The BP The BP network was trained with the original input sample P.Two new training samples P1 and P2 were obtained by adding and reducing 10% of the original values of one target characteristic variable while keeping the others constant.P1 and P2 were used as the simulation samples to perform simulation by the network established with (1), and two simulation results A1 and A2 were produced.The difference between A1 and A2 was calculated to obtain the impact value of this characteristic variable on the model output.The MIV was calculated based on the existing sample size, that is, the MIV value of this characteristic variable.Steps (2)–(5) were repeated to sequentially calculate the MIV of each characteristic variable in the sample P.th independent variable and m is the number of independent variables in the sample P. Moreover, The weight of the impact of each characteristic variable was calculated on the prediction result using the following Equation (1):than 85% .Excessive input parameters or redundancy can increase network complexity and even reduce accuracy in modeling. Therefore, the characteristic parameters mentioned in The BP neural network is the most widely applied algorithm among artificial neural networks. However, it has some disadvantages, such as slow learning-convergent velocity, difficulty in network structure determination, and inability to obtain accurate initial connection weights and thresholds. This study adopted the multiple population genetic algorithm (MPGA) to optimize the neural networks in an attempt to compensate for the aforementioned drawbacks of BP neural network .The single population genetic evolutionary framework of GA was broken, and multiple populations were introduced to perform optimization search simultaneously. The control parameters were different for each population.Various populations were connected by immigration operators, and multiple populations co-evolved.The artificial selection operators were used to save the best individuals of each population in each generation, and the best individual and the least preserved generation was regarded as the criterion for terminating the algorithm.The genetic algorithm (GA) is a random and self-adaptive search algorithm for global optimization developed by borrowing the ideas of natural selection and a random evolutionary mechanism . It doesBinary coding. Each weight and threshold were binary coded. The codes of all the weights and thresholds comprised the code for an individual; multiple individuals formed a population; and multiple populations were initialized in parallel.Fitness calculation. For the multiple populations of (1), the fitness function was substituted to calculate the fitness value of each individual in each population.Selection, crossover, and mutation. The selection operators were used for selection, and the crossover probability and mutation probability were used to generate offspring.Fitness calculation for offspring.Immigration operations. The best individuals in the population were identified to perform immigration operations.Artificial selection. The best individual of each population was collected by artificial selection operator and put into an elite population for preservation.The best individual in the elite population was found, and the number of preservation generations for this best individual was updated. When the number of preservation generations met the minimum preservation generation, the algorithm was terminated, and the optimal individual was decoded to generate the optimal parameters.The algorithm flow chart of MPGA in optimizing BP neural network is shown in A big variation exists between the blood pressure and the corresponding waveform characteristics of an individual. In the current circumstances, this study only considered two characteristics of individuals, BMI and gender, which were definitely not sufficient. Moreover, to reduce overfitting, this study trained multiple independent blood-pressure models and integrated the models using different proportional coefficient set for different individuals, achieving continuous monitoring of individualized blood pressure. In this study, MPGA was used to determine the proportional coefficients of these models, and finally to get the individualized blood-pressure monitoring models.The detailed information of the experimental data is shown in The distribution of blood pressure obtained from the experiment is shown in Based on the nature of the wavelet basis and the ECG signals that need to be processed, “coif5” was used as the wavelet packet base. The number of decomposition layers was set to 3, and “SURE entropy” was adopted as the information cost function to select the best wavelet base. The “ddencmp” function was applied to acquire the denoising threshold according to the nature of the signal. Then, the wavelet coefficients corresponding to the optimal wavelet basis were quantized, and finally the denoised signals were obtained via the reconstruction algorithm. The denoising results are shown in For the PPG signals, the initial denoising and processing were similar to the ECG signals. Nevertheless, for the baseline drift with great influence, this study treated it further using the cubic spline interpolation method . The PPGThe BP neural network used in this study was based on the default Neural Network Toolbox of MATLAB 2016b. The MPGA was based on the Sheffield Genetic Algorithm Toolbox. The data from 17 individuals, Test 1–Test 8 and Test 14–Test 22, a total of 775 sets of data, were disordered first and used for the basic blood-pressure model construction. The data from the other 10 individuals, Test 9–Test 13 and Test 23–Test 27, a total of 120 sets of data, were used for the individual’s blood-pressure model construction and testing.A three-layer BP neural network can implement arbitrary non-linear mapping if the number of nodes in the hidden layer is not limited. This study selected a single hidden layer BP neural network to improve the training speed and simplify the neural network structure. It was applied to the 775 sets of data to construct basic models. Then, the 24 parameters, including the waveform characteristic parameters and the individual’s features, were used as input, and the corresponding SBP and DBP values measured by the sphygmomanometer were used as output to construct one BP blood-pressure model regarding SBP (nets0) and one BP blood-pressure model regarding DBP (netd0).m is the number of nodes in the hidden layer, n is the number of nodes in the input layer, l is the number of nodes in the output layer, and m only needs to be set within the range. In this study, it was set to 11.The BP neural network parameters were set as follows: The learning rate was 0.01, the activation function of the hidden layer was “tansig”, the activation function of the output layer was “purelin”, and the learning algorithm was the Levenberg–Marquardt algorithm with fast convergence, which was the “Trainlm”. With respect to the setting of the number of nodes in the hidden layer, the following empirical equation was used (Equation (2)):ayer was ,16. As tc, PTTa, HR, tbf, tfb, tae, tdown, tup, tbfr, tfbr, tupr, tdownr, taer, Her, Hfr, and K; and for DBP were BMI, PTTc, PTTa, HR, tbf, tfb, tae, tdown, tup, tbfr, tfbr, tupr, tdownr, taer, Har, Her, Hgr, and K. The second round was based on the results of the first round. The orders of the MIV values of the input features of the SBP and DBP models are shown in downr, tbf, tfbr, PTTc, tup, tbfr, tdown, Her, taer, tupr, HR, K, and PTTa) and14 for DBP .Based on the BP neural network nets0 and netd0 constructed in In addition to the input characteristics identified in The RMSE values for SBP and DBP of the BP neural network for various nodes of the hidden layer are shown in The parameter setting of MPGA in optimizing the BP neural network was as follows: The range for weights and threshold of the network was −0.5–0.8; the individual number in an initial population was 20, the binary digit for an individual was 10, the population number was 5, the preservation generation for the best individual was 3, the crossover probability was randomly generated within the range of 0.7–0.9 the mutation probability was randomly generated within the range of 0.001–0.05 and the output of the target function was network RMSE. Based on the 775 sets of data for the basic model construction, 5-CV was applied to train 5 independent SBP networks  and 5 independent DBP networks . The evolution curve of Nets1 is shown in For the same data samples (training set and test set), the RMSE values before and after optimization are shown in The continuous blood-pressure monitoring models for an individual could be obtained using the networks trained as in The RMSE, mean absolute deviation (MAD) (Equation (6)), and standard deviation (STD) (Equation (7)) were used to evaluate the model prediction capability. For the individualized models from This study constructed partial least squares regression (PLSR) models based on the same data to evaluate the validity of the models objectively. PLSR is a modeling method that considers both extracting the principal components of the dependent and independent variables and maximizing the correlations between the extracted principal components in the regression process ,43. The This study introduced the Pearson correlation coefficient (denoted as R) analysis , as wellIn this study, a set of non-invasive continuous blood-pressure monitoring models based on photoplethysmography and electrocardiography was proposed. Compared with the traditional PWTT-based blood-pressure estimation models, more characteristics  were introduced in the parameter selection in model construction, including much more abundant information related to blood pressure. The characteristic parameters were screened so that only the features with large impacts on the model output were included to reduce information redundancy, thus reducing the complexity of the network structure and improving the generalization ability of the network. In view of the disadvantage that the initial connection weights and thresholds that have a great influence on the network cannot be obtained accurately, the MPGA was adopted to determine the initial weights and thresholds of the neural network, which greatly improves the blood-pressure prediction performance of the neural network. Model integration was performed regarding the multiple-trained independent models with various proportional coefficients to reduce overfitting and achieve individualized long-term blood-pressure monitoring. The models in this study were proposed for the prediction of blood pressure over a longer period of time. And the models validation and testing based on the 12 sets of blood-pressure data collected in 20 min showed that the models could be used for long-term blood-pressure monitoring.By contrast with Tan’s research , the optAlthough the proposed models in this study achieved a certain prediction accuracy, the following issues remained. First, the experiment sample size was small, and all the participants were healthy people. The diversity of experimental data needed to be further expanded. Second, the continuous data collection time of each experiment was limited to 20 min, and the obtained continuous blood-pressure data were only up to 12 data sets. Therefore, the blood-pressure monitoring verification could only be performed for a period of 20 min but not longer, such as half a day, 1 day, or even 1 week. In addition, regarding the characteristics of individuals, this study only considered gender and BMI, while these two characteristics were definitely not sufficient keeping in mind the differences in blood pressure among individuals. Finally, the proportional coefficients need to be calibrated during model integration for different individuals, and the calibration time for a single individual’s blood-pressure models was approximately 1 min.The MPGA–MIV–BP monitoring models proposed in this study used MIV to reduce feature redundancy and simplify the model structure. Furthermore, MPGA was used to optimize the initial weights and thresholds of the BP neural network, and determine the proportional coefficients for model integration, effectively reducing overfitting and improving the prediction accuracy of SBP and DBP. The MPGA–MIV–BP monitoring models performed well during long-term (20 min) blood-pressure monitoring , providing a direction for the clinical application of non-invasive continuous blood-pressure monitoring equipment.


The time a patient spends with blood pressure at target level is an intuitive measure of successful BP management, but population studies on its effectiveness are as yet unavailable.We identified a population-based cohort of 169,082 individuals with newly identified high blood pressure who were free of cardiovascular disease from January 1997 to March 2010. We used 1.64 million clinical blood pressure readings to calculate the TIme at TaRgEt (TITRE) based on current target blood pressure levels.The median (Inter-quartile range) TITRE among all patients was 2.8  months per year, only 1077 (0.6%) patients had a TITRE ≥11 months. Compared to people with a 0% TITRE, patients with a TITRE of 3–5.9 months, and 6–8.9 months had 75% and 78% lower odds of the composite of cardiovascular death, myocardial infarction and stroke  and 0.22 , respectively). These associations were consistent for heart failure and any cardiovascular disease and death , among people who did or did not have blood pressure ‘controlled’ on a single occasion during the first year of follow-up, and across groups defined by number of follow-up BP measure categories.Based on the current frequency of measurement of blood pressure this study suggests that few newly hypertensive patients sustained a complete, year-round on target blood pressure over time. The inverse associations between a higher TITRE and lower risk of incident cardiovascular diseases were independent of widely-used blood pressure ‘control’ indicators. Randomized trials are required to evaluate interventions to increase a person’s time spent at blood pressure target. Long-term lifestyle and pharmacological interventions to lower blood pressure (BP) have been shown in randomized trials to be effective in reducing cardiovascular morbidity and mortality–5but litThe ‘area under the time curve’ of BP level is associated with cardiovascular disease endpoints. The concWe studied an English population because of national remuneration scheme to achieve the same BP lowering goal across general practices. and becaThe authors declare that all methods are available within the manuscript (and its online-only Supporting Information).www.caliberresearch.org/portal). The study was approved by the Independent Scientific Advisory Committee of the Medicines and Healthcare products Regulatory Agency in accordance with the Declaration of Helsinki.We used primary care records of individual patients from the 225 primary care practices contributing data to the Clinical research using LInked Bespoke studies and Electronic health Records (CALIBER) program during the study period from January 1997 to March 2010. CALIBER links patient records from four different data sources: primary care , disease registry , hospital care , and death registry . A description of CALIBER,24,26,27Newly identified high BP was defined based on recorded values in primary care according to the most recent guidelines: ≥140 mmHg systolic blood pressure (or ≥150 mmHg for people aged ≥60 years without diabetes and chronic kidney disease) and/or ≥90 mmHg diastolic blood pressure. The majority of general practitioners use digital sphygmomanometers, which haThe main exposure was the annual percent time BP at management target, averaged over follow-up years (in short: TIme at TaRgEt (TITRE)). The treatment goal was set to <140/90 mmHg or <150/90 mmHg for patients aged ≥60 years without diabetes and chronic kidney disease according to treatment guidelines.–31 TITREAt baseline we evaluated systolic and diastolic BP and stage-two hypertension status (systolic BP ≥160 mmHg or diastolic blood pressure ≥100 mmHg). On follow-up we evaluated lifestyle intervention: receiving diet advice or regimen , and smoking cessation among people who reported at baseline as current or past smoking. Pharmaceutical interventions included first BP-lowering drug, the delay from study entry to prescription of first BP-lowering drug, snapshot control status defined as having a single on-target blood pressure measure within the first year of follow-up, and a binary follow-up control status defined as the mean follow-up BP below or exceeding the treatment target. We evaluated in sensitivity analyses other alternative BP control measures, such as the median absolute deviation for average BP (7.5 mmHg for SBP and 5 mmHg for DBP), BP visit-to-visit variability, and number of follow-up BP measures.2, based on the Modification of Diet in Renal Disease formula of 0.47 , 0.25, 0.22 and 0.26, respectively . A higheFor secondary endpoints, the graded and stepwise associations were observed for TITRE and incident stable angina, peripheral artery disease, and all-cause mortality were also observed. For example, individuals with a TITRE of <3 months, 3–5.9 months, 6–8.9 months, and 9–11.9 months had multiple adjusted odds ratio [OR] of stable angina of 0.56 , 0.34 , 0.24  and 0.16 , respectively. The risk of multicollinearity in study models was small, the area under the ROC curve of study models ranged from 0.81, 0.90, indicating good fit to the data, and Akaike’s information criteria suggested good prognostic value of TITRE .In a hypothetical scenario, if patients experienced the standardized event risk of one level higher TITRE category than their own, in every 100,000 hypertensive individuals in the UK, 1824 deaths and cardiovascular disease events might be prevented or deferred over five years. In this hypothetical scenario, the greatest number of events prevented would be for patients with 0% or missing TITRE who, with TITRE of <3 months, would see an estimated 1990 deaths and cardiovascular diseases prevented or deferred, including 175 cardiovascular deaths, myocardial infarctions and strokes and 153 cases of incident heart failure .Unadjusted event risks were similar in people who either achieved or did not achieve snapshot ‘control’ during the first year of follow-up . A consiWe report the first study investigating the time profiles of BP at target in usual care and subsequent risks of death and incident cardiovascular diseases. In 169,082 people with newly identified high BP, and 1.64 million BP measurements over a median of nearly five years, we estimated that BP was at current target levels for less than three months per year in half of all cases. Compared to a 0% TITRE, a TITRE based on current guideline recommendation (3–6 months) was assoA key finding from our study was how often blood pressure is measured in the 4.9 years after newly diagnosed hypertension. We found a median of 7 measurements per patient and this translated into a median of 1.6 measures per year. Clearly a more accurate measurement of TITRE would be made if BP measurements were more frequent; the new evidence we provide here is that even at this frequency of measurement it is possible to distinguish clinically relevant (in terms of prognosis) groups based on TITRE.Clinical guidelines and many previous observational studies have defined ‘BP control’ as a binary variable, based on single cross-sectional BP measurement,35 aboveA higher TITRE was associated with a number of factors, including; achieving BP control at a single time point within the year, and guideline-recommended intervention such as initial prescription of thiazides for BP lowering and provision of dietary advice. It is worth noting that, half of the hypertensive patients were not prescribed BP-lowering medications, suggesting substantial variation in hypertension management with usual care, combining lifestyle intervention, blood pressure monitoring and pharmaceutical intervention. The adherence to treatment strategies has also been shown to influence the management of blood pressure. This conA higher TITRE was associated with lower cardiovascular mortality and morbidity; furthermore, lower TITRE had a stronger association with risk of cardiovascular outcomes. The gradient of risk of cardiovascular diseases across different TITRE categories demonstrates not only its capability to better quantify the attributable risk to differences in longitudinal BP management, but also its potential to better characterize the benefits of BP-lowering interventions in reducing cardiovascular risk and mortality. The gradient of risk associated with TITRE remains after taking account of other BP management indicators, and in strata defined by antihypertensive drug use  and crosIt is worth noting that achieving a high TITRE at current BP targets was associated with relative risk reductions  greater than reported in a recent meta-analysis of 19 randomized trials of intensive blood pressure lowering medication, which reported a 14% reduction in a composite of major cardiovascular events, but no clear effect on heart failure. This conOur study showed that the effects of TITRE on cardiovascular outcomes were greater and more informative than more commonly advocated measures of BP control. Thus snapshot measures cannot readily detect episodic hypertension in which systolic blood pressure oscillates above and below targets, and commClinicians and policymakers face choices between widely different strategies for managing BP. Our study suggests that before considering more aggressive treatment and lower BP targets, it might be more effective to focus on achieving, and maintaining BP control using existing blood pressure targets—a goal that is not achieved in US, Europe, or in low and middle income countries.–9,39–41 There is currently no mention in US, EuropeanImplementation of the TITRE is feasible in electronic health records systems used to monitor clinical care and by patients themselves using home BP monitoring and apps. It could be further accompanied by visualizations of the time profile and decision support to target BP-lowering management.,44Our findings suggest the need for international comparative study on TITRE and its association with cardiovascular risk in other health care system with different strategies in hypertension management (for example. different pattern of hypertensive medication use), and pragmatic real-world randomised trials evaluating interventions  to increase the time spent at BP target, the best strategies to measure time at target  and their impact on the incidence of cardiovascular diseases. In considering such trials and treatment guidelines, future research is required to evaluate whether strategies aimed at achieving a 20 mmHg lower BP target is more effective than strategies aimed at extending TITRE by 3 or more months.The strengths of this study lie in this large nationally representative population from a country which incentivizes family physicians to meet the same national BP targets. CALIBER’Almost all newly identified individuals with high BP fail to achieve and sustain consistent blood pressure control. Higher time at target (TITRE) was associated with lower risk of incident cardiovascular diseases and was an effective predictor of clinical outcomes, with a high clinical relevance and actionability. Efforts to lower cardiovascular risk among hypertensive patients should fully utilize multiple measurements of BP in usual care or via self-monitoring, with treatment strategies to attain a high TITRE.S1 Method(DOCX)Click here for additional data file.S2 Method(DOCX)Click here for additional data file.S3 Method(DOCX)Click here for additional data file.S4 Method(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.


Children with chronic kidney disease (CKD) are at high risk of anemia, an important risk factor for cardiovascular disease and poor quality of life. The present study used baseline data from the Korean cohort study for Outcome in patients With Pediatric Chronic Kidney Disease (KNOW-PedCKD). A Total of 437 patients was included in the analyses excluding missing data. The characteristics of patients with and without anemia and those of patients with and without iron deficiency were compared. Logistic regression analysis and Pearson correlation were conducted to evaluate associated risk factors and correlations in children with CKD. Anemia in children with CKD was associated with older age, low body weight and body mass index (BMI) z-score, birth age, preceding glomerulonephritis, decreased estimated glomerular filtration rate (eGFR), low levels of serum albumin and calcium, high levels of serum intact parathyroid hormone (iPTH), and serum phosphorus. Anemia was correlated positively with changes in the BMI z-score, body weight, and serum albumin and cholesterol levels, but correlated negatively with serum calcium, iPTH, ferritin levels, and transferrin saturation. Iron deficiency in children with CKD was associated with young age, low hemoglobin and serum ferritin levels, high BMI z-scores, and low levels of serum iPTH. This is the first nationwide cohort study of anemia in Korean children with CKD and the first prospective pediatric CKD cohort study in Asia. The study results demonstrated that anemia and iron deficiency are affected by various factors, including age, BMI, and levels of serum iPTH. To improve the retrospective outcome of affected children, it is important to understand the effect of each of these factors and to attempt an early intervention to prevent anemia and iron deficiency by regular measurement of these parameters in children at risk. Anemia is a major complication in children with chronic kidney disease (CKD). Data from the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS) revealed the prevalence of anemia in children with CKD, ranging from 73 to 93%, depending on the CKD stage . Among cThe chronic inflammatory state in patients with CKD results in decreased erythropoiesis in the bone marrow, reduced production of erythropoietin (EPO) in the kidneys, and impaired iron absorption and mobilization due to increased production of hepcidin in the liver. Uremia, oxidative stress, and nutritional deficiencies also contribute to anemia in CKD . TherefoThe objectives of this study were to examine the characteristics of patients with anemia and iron deficiency among children with CKD and to identify associated risk factors and correlations using baseline data from the Korean cohort study for Outcome in patients With Pediatric Chronic Kidney Disease (KNOW-PedCKD).n = 437). A total of 437 patients was included in the analysis. If the individual tests have missing data, they were excluded in the final analysis. The KNOW-PedCKD study is a nationwide observational prospective cohort study of pediatric CKD patients with CKD stages 1–5 recruited from seven major pediatric nephrology centers in Korea. Details on the study design and objectives have been published previously . For theCKD was defined and staged according to the Kidney Disease Improving Global Outcomes (KDIGO) criteria . For patr correlation. All statistical analyses were performed using SAS statistical software .Categorical variables expressed as percentages were compared between groups with the Chi-square test and Fisher’s exact test. Continuous variables were expressed as a mean ± standard deviation and analyzed by the Kruskal–Wallis test. A logistic regression analysis was conducted to determine the odds ratios (ORs) and confidence intervals (CIs) of associated risk factors for anemia and iron deficiency in the cohort. The correlations among characteristics of anemia in the cohort were analyzed with a Pearson n = 299 versus n = 138). The most common disease leading to CKD was renal hypoplasia (41.4%), while 66 patients had kidney dysplasia with/out vesico-ureteral reflux (VUR) (15.1%), and 27.7% had glomerulonephritis. Overall, 27.2% of patients were in CKD stage 2, while a minority (3.9%) had CKD stage 5 in a pre-dialysis state.The demographic characteristics and clinical manifestations of patients with CKD are shown in p < 0.001), glomerulonephritis as a preceding disease (p < 0.001), higher CKD stages (p < 0.001), higher diastolic blood pressure (DBP) (p = 0.029), lower body weight (p = 0.036), proteinuria (p < 0.001), and use of a renin-angiotensin system inhibitor (p = 0.002) were significantly associated with CKD patients with anemia, compared to those without. CKD patients with anemia were treated with erythropoietin (EPO)-stimulating agents  and supplemental iron therapy , and showed significantly lower ferritin levels (p < 0.001) than those without anemia. There were no significant differences in sex distribution, platelet counts, systolic blood pressure, or transferrin saturation according to the presence of anemia.Characteristics of the CKD patients with and without anemia are listed in p < 0.001) (p = 0.003).Patients with CKD showed a bimodal distribution with low hemoglobin levels at ages 0–2 years (11.14 ± 1.64 g/dL) and at ages 6–12 years (11.65 ± 1.79 g/dL) . Hemoglo< 0.001) . Hemoglop < 0.001), low weight , low body mass index (BMI) z-score , existence of glomerulonephritis , high levels of ionized parathyroid hormone (iPTH)  and serum phosphorus , low serum albumin  and calcium  levels, and low eGFR  in the univariate logistic regression. The multiple logistic regression analysis revealed the existence of glomerulonephritis  to be an independent risk factor associated with anemia in children with CKD , weight , serum albumin , and cholesterol , but correlated negatively with serum calcium , iPTH , ferritin , and transferrin saturation  (Anemia correlated positively with changes in BMI z-score (< 0.001) .p = 0.002), existence of glomerulonephritis as a preceding disease (p = 0.017), systolic blood pressure (p = 0.036), and presence of comorbidities (p = 0.009) were significantly higher in patients with iron deficiency than in those without. Other variables, including sex, weight, CKD stages, and use of iron supplementation agents, did not differ between the two groups.Characteristics of the CKD patients with iron deficiency are summarized in p < 0.001); however, the lowest age group with transferrin saturation was 0–2 years  (p < 0.001). In contrast, there were no significant differences in transferrin saturation across the CKD stages (p < 0.001). Transferrin saturation showed a significantly lower percentage in the congenital, hereditary, cystic disease group .In CKD patients with iron deficiency, ferritin levels were significantly lower in the 2–6 year-old age group  . InteresD stages . As showp < 0.001), lower hemoglobin , and lower ferritin  levels in the univariate logistic regression analysis. The multiple logistic regression analysis demonstrated that an increased BMI z-score  and decreased iPTH , hemoglobin , and ferritin  levels were risk factors associated with iron deficiency in children with CKD  pathway to reduce proteinuria in glomerulonephritis modulate erythropoiesis by regulation of angiotensin II signaling. Angiotensin II is involved in the proliferation and differentiation of hematopoietic cell types, especially those of red blood cell lineage. Both angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) induce adverse effects related to decreased hematocrits . NotwithFerritin increased as the CKD stage increased in our cohort. According to another report, ferritin itself could act as an inflammatory marker . In addir = 0.324, p < 0.001 and r = 0.434, p < 0.001, respectively, The correlation results in our study indicated that anemia was positively correlated with low body weight and BMI z-score (Hypertension is one of the major causes of CKD worldwide . Our resAnemia and iron deficiency are associated risk factors of CKD. Despite the established treatment of anemia and iron deficiency in CKD patients, our result suggests the importance of simultaneous use of EPO-stimulating agents and iron supplements as an effective treatment approach.There are several limitations in this study. First, there could be unavailable data or information of patients of the CKD cohort. Second, we could not include the patients’ diets and calorie intake in this report. Third, we may not have excluded potential significant inter-study variations in the study setting. In addition, it is not clear in CKD patients as to whether these indicators are the effect of CKD itself or impact of anemia. We plan in future studies to examine the relationship between the impact of anemia on quality of life, cognitive function, exercise capacity, and cardiovascular function and anemia in the KNOW-pedCKD cohort.This article is noteworthy because it is the first multicenter cohort study investigating the impact of anemia in Korean pediatric patients with CKD and the first prospective pediatric CKD cohort study in Asia. Supported by clinical evidence, our results indicate that anemia and iron deficiency are key factors associated with CKD in pediatric patients. Understanding the multiple risk factors causing anemia in CKD will improve the outcomes of children with CKD. Furthermore, ESA and iron-replacement therapy have revolutionized the treatment of anemia in children and the negative effects of anemia can be improved by these treatments. Additional studies are necessary to address the issue of anemia in pediatric CKD according to each patient’s diet or quality of life in the future.


The index for a target that can lead to improved prognoses and more reliable therapy in each heterogeneous patient with heart failure with preserved ejection fraction (HFpEF) remains to be defined. We examined the heterogeneity in the cardiac performance of patients with HFpEF by clarifying the relationship between the indices of left atrial (LA) volume (LAV) overload and pressure overload with echocardiography. We enrolled patients with HFpEF (N = 105) who underwent transthoracic echocardiography during stable sinus rhythm. Relative LAV overload was evaluated using the LAV index or stroke volume (SV)/LAV ratio. Relative LA pressure overload was estimated using E/e’ or the afterload-integrated index of left ventricular (LV) diastolic function: diastolic elastance (Ed)/arterial elastance (Ea) ratio = (E/e’)/(0.9 × systolic blood pressure). The logarithmic value of the N-terminal pro-brain natriuretic peptide was associated with SV/LAV . The pulmonary capillary wedge pressure was positively correlated to Ed/Ea . SV/LAV was negatively correlated to Ed/Ea , with no observed between-sex differences. The correlations between the LAV index and E/e’ and Ed/Ea and between SV/LAV and E/e’ were less prominent than the abovementioned relationships. SV/LAV and Ed/Ea, showing relative LAV and LA pressure respectively, were significantly but modestly correlated in patients with HFpEF. There may be considerable scatter in the relationships between these indices, which could possibly affect the selection of medications or efforts to improve the prognoses of patients with HFpEF. However, the index for a target that can lead to improved prognoses and more reliable therapy in a heterogeneous patient population remains to be defined. Early works have suggested that E/e’ could be used to reliably estimate the left ventricular (LV) filling pressure in the clinical setting of diastolic heart failure (HF)5. The correlations between E/e’ and direct left atrial (LA) pressure or pulmonary capillary wedge pressure (PCWP) are significant in a stable state8. LV diastolic elastance, expressed as Ed = (E/e’)/stroke volume (SV)9 or (E/e’)/LV end-diastolic volume10, and arterial elastance, expressed as Ea = (0.9 × systolic blood pressure)/SV9, are both higher in women than in men under stable conditions10. We previously reported that the Ed/Ea ratio is an index of LV diastolic function relative to afterload and is calculated as (E/e’)/(0.9 × systolic blood pressure), when Ed = (E/e’)/SV12. Therefore, Ed/Ea is not calculated by the parameters of cardiac volume, such as LA volume (LAV) and SV.The heterogeneity in the cardiac structure and function of patients with heart failure with preserved ejection fraction (HFpEF) is well known13. In this study, we examined the extent to which the echocardiographic indices of volume and pressure overload in the left atrium are correlated with each other and the associated sex differences to elucidate the appropriate therapy for patients with HFpEF who have heterogeneous cardiac performance; these relationships could possibly affect the selection of medications or efforts to improve the prognoses of these patients. The LAV index (LAVI) or SV/LAV ratio was used as a marker of LAV overload, and the E/e’ or Ed/Ea ratio was used as a marker of LA pressure overload. As markers of myocardial function, we used the N-terminal pro-brain natriuretic peptide (NT-proBNP) levels and PCWP.We recently reported that larger LAVs, relatively smaller LV volumes, and higher E/e’ and Ed/Ea ratios were observed in elderly women than in men with preserved LV ejection fraction (LVEF), in both those with and without HF13. The PURSUIT HFpEF registry is from a prospective, multicentre observational study in which collaborating hospitals in the Osaka region of Japan record the clinical, echocardiographic, and outcome data of patients with HFpEF (UMIN-CTR ID: UMIN000021831). This study complied with the tenets of the Declaration of Helsinki, and the protocol  was approved by the ethics committee of each participating hospital . Briefly, hospitalized patients with HF and an LVEF ≥ 50% were prospectively registered. All methods were performed in accordance with the relevant guidelines and regulations. All patients provided written informed consent to participate. We excluded patients with atrial fibrillation and/or considerable mitral or aortic valve disease.We enrolled patients with HFpEF  recruited from the PURSUIT HFpEF  registry15. In patients with HFpEF, echocardiography was performed while the patient was in stable condition before discharge. Volumetry was standardized using a modified Simpson’s method. The LAVI was calculated as the LAV divided by the body surface area. As markers of diastolic stiffness estimating the relative LA pressure overload, E/e’ and Ed/Ea (afterload-integrated diastolic elastance) were examined16. We evaluated the LAVI and LA ejection fraction (LAEF), which was calculated using SV/LAV, as relative markers of LAV overload. To ensure highly accurate measurements from the echocardiographic data, we performed short-course training sessions for echo technicians in each participating hospital several times13. The serum level of NT-proBNP and the estimated glomerular filtration rate (eGFR) were examined at the same time. A subset of the patients (N = 46) underwent right-heart catheterization and were examined for their PCWPs before discharge. The correlations among laboratory data, PCWP, and echocardiographic parameters were evaluated.Echocardiographic measurements were obtained according to the criteria of the American or European Society of Echocardiography during stable sinus rhythmContinuous variables were expressed as means ± standard deviations, and categorical variables were presented as frequencies and percentages. The differences in categorical variables between the groups were compared using the chi-square test, and the differences in continuous variables between the groups were compared with Student’s t-test or Welch’s t-test, as appropriate. The correlations were tested using Pearson or Spearman coefficients, and the p-values were examined using regression analysis. Between-sex differences were evaluated using an interaction analysis. A p-value < 0.05 was considered significant.The clinical and laboratory characteristics and echocardiographic data of the patients are shown in Table The logarithmic value of NT-proBNP was modestly associated with SV/LAV  but not with LAVI , E/e’ , or Ed/Ea  /(0.9 × systolic blood pressure) and the LV dimension and LVEF are within the normal ranges for patients with HFpEF, the relationship between SV/LAV and Ed/Ea may indirectly reflect ventriculo-arterial coupling. Third, we could not exclude the potential for measurement bias among facilities or investigators because the echocardiography evaluations were performed locally. Finally, we could not discuss the echocardiographic parameters of patients with atrial fibrillation.The SV/LAV and Ed/Ea ratios, which represent volume and pressure in the left atrium, were significantly but modestly correlated in patients with HFpEF. The considerable scatter in the relationship may affect the selection of medications or efforts to improve the prognoses of patients with HFpEF.


Pneumocystis jirovecii pneumonia (PCP) remains an important cause of morbidity and mortality in kidney transplant recipients. While the acute phase toxicity in patients with PCP is well-characterized, there is a lack of data on the effects of PCP on long-term graft outcome.This retrospective observational study analyzed 1502 adult patients who underwent kidney transplantation at Seoul National University Hospital between 2000 and 2017. After a propensity score matching was performed, the graft and survival outcomes were compared between PCP-negative and PCP-positive groups.P = 0.027] and mortality  than the PCP-negative group. However, the PCP event was not related with subsequent development of de novo donor-specific antibodies or pathologic findings, such as T-cell or antibody mediated rejection and interstitial fibrosis and tubular atrophy.A total of 68 patients (4.5%) developed PCP after transplantation. The multivariable Cox analysis showed that positivity for cytomegalovirus and lack of initial oral antibiotic prophylaxis were risk factors of post-transplant PCP. The PCP-positive group had higher hazard ratios of graft failure [adjusted hazard ratio (HR), 3.1 (1.14–8.26); PCP is a risk factor of long-term graft failure and mortality, irrespective of rejection. Accordingly, appropriate prophylaxis and treatment is needed to avoid adverse transplant outcomes of PCP.The online version of this article (10.1186/s12882-019-1407-x) contains supplementary material, which is available to authorized users. With the continued development of immunosuppressive regimens in the past decade, death-censored graft failures have gradually decreased in recipients of both living and deceased kidney recipients , 2. HoweInfection is an important factor in relation to the risk of death in kidney transplant recipients, and the second most common cause of death after cardiovascular disease in patients with functioning grafts –5. The iPneumocystis jirovecii is an opportunistic pathogen that causes severe pulmonary infection in immunocompromized hosts [P. jirovecii pneumonia (PCP) varies from 0.6 to 14% among kidney transplant recipients without prophylaxis, with a mortality of up to 50% despite aggressive antibiotic therapy [ed hosts . The inc therapy , 9. Seve therapy , but the therapy –14. Thisn = 260) or who received simultaneous kidney–pancreas or kidney–liver transplants (n = 65) were excluded. Consequently, the remaining 1502 patients were finally included and their data were reviewed. The requirement of informed consent was waived by the board.The study design was approved by the institutional review board of Seoul National University Hospital (no. H-1805-173-948) and complied with the Declaration of Helsinki. This retrospective observational study included total 1827 patients who had kidney transplantation at Seoul National University Hospital from January 2000 to December 2017. Patients who were under 18 years old  and human leukocyte antigen (HLA) typing were collected to evaluate ABO compatibility and the number of HLA mismatches. Immunosuppressive therapy was determined, including induction therapy with basiliximab or anti-thymocyte globulin, and calcineurin inhibitors for maintenance therapy. A combination of steroids, mycophenolic acid, and calcineurin inhibitors was the standard maintenance immunosuppressive regimen in our center. PCP was defined as the presence of findings suspicious of PCP detected by a radiologist on chest computed tomography combined with PCP positivity on polymerase chain reaction or direct immunofluorescence stain of sputum or bronchoalveolar lavage fluid. Initial use of oral prophylactic antibiotics was defined as a prescription of trimethoprim/sulfamethoxazole for more than 4 weeks during the first month after kidney transplantation. There were no cases with pentamidin or atovaquon for prophylaxis. CMV positivity was defined as > 20 copies/ml on polymerase chain reaction or positive result on viral culture, according to the definition of CMV infection .The primary outcome was death-censored graft failure defined as a return to dialysis or kidney re-transplantation. The secondary outcome was all-cause mortality, based on data obtained from the National Database of Statistics Korea. All patients were followed until graft failure or March 2018. Data on biopsy-proven acute T-cell-mediated rejection, acute antibody-mediated rejection, and interstitial fibrosis and tubular atrophy were also collected. Protocol biopsies were performed at zero-time (postreperfusion), on the 10th day and 1 year after transplantation, and every year thereafter. Additional kidney biopsies were performed if graft function deteriorated or any suspicious symptoms or signs of rejection were observed. All pathologic findings were examined by a nephropathologist. The development of de novo donor-specific antibody (DSA) was defined as any newly developed anti-HLA class I or II antibody.2 and Fisher’s exact tests and continuous variables were compared using Student’s t-test. Risk factors for PCP occurrence were analyzed using univariable and multivariable Cox proportional hazard models.All statistical analyses were performed using Stata software . Data are expressed as mean ± standard deviation for continuous variables or counts with percentages for categorical variables. Among the baseline characteristics, categorical variables were compared using χP values under 0.1 in multivariable Cox analysis for the risk of PCP. Then, the cases were matched on propensity score in a 1:2 block, using a nearest neighbor matching algorithm with replacement, using the statistical package psmatch2. Following propensity score matching, graft survival, overall patient survival, risk of rejection and development of DSA were analyzed using univariable and multivariable Cox proportional hazard models. Because PCP infection was a time-dependent covariant in the Cox model, we used the stssplit function in Stata to split the time at which PCP occurred. The proportionality assumption was checked for proportional hazard Cox regression. Survival curves were drawn using the Kaplan–Meier method, with comparisons between groups carried out using the log-rank test. A P value < 0.05 was considered to indicate statistical significance.Propensity score matching was performed to account for the imbalance in baseline characteristics between the PCP-positive and -negative groups. Scores were created with matching variables including age, gender, donor type, type of pre-transplant dialysis, hypertension, the usage of prophylactic antibiotics, and CMV positivity which had Table P = 0.008). Cox regression analysis considering PCP as a time-dependent variable was subsequently performed. Both univariable and multivariable analyses showed a significant relationship between PCP and graft failure, with HRs of 3.34 (1.31–8.56) (P = 0.012) and 3.33 (1.30–8.53) (P = 0.012), respectively. Although the acute T cell-mediated and antibody-mediated rejection episodes or other immunological findings such as interstitial fibrosis and tubular atrophy and de novo DSA were additionally adjusted, the PCP-positive recipients had a higher risk of death-censored graft failure than the PCP-negative recipients ; P = 0.027).We performed propensity score matching to mitigate the difference in baseline characteristics between the PCP-positive and -negative groups. Table n = 9) are shown in Additional file The clinical information on the PCP-positive patients with graft failure  and 13 patients (11.0%) died in the PCP-positive and -negative groups, respectively. The 28 PCP-positive patients had concurrent other infectious diseases, and 8 patients died (28.6%). All-cause mortality was higher in the PCP-positive group than in the PCP-negative group  patients died within 3 months after diagnosis of PCP infection. The causes of death in these patients were either PCP itself or the other infection superimposed on PCP. Thus, considering both the significant effect of PCP on mortality and the negative effect on graft outcome over long-term period, prophylaxis against PCP may be strongly recommended. According to the Kidney Disease Improving Global Outcomes guideline, it is recommended that all recipients receive prophylaxis against PCP for 3–6 months after transplantation . HoweverAlthough our data are informative, there are some limitations. First, it was a retrospective study, making it difficult to demonstrate cause and effect definitively. Nevertheless, the study achieved its primary purpose by demonstrating the relationship between PCP and transplant outcomes. Second, the current definition of prophylactic antibiotics was 1 month, which was shorter than the guideline recommendation , which might increase overall risk of PCP in study subjects. The duration of antibiotic prophylaxis  might differ between centers, which could also alter the risk of PCP or other transplant outcomes. However, this did not hinder the study purpose on the relationship between PCP and transplant outcomes. Lastly, the present observational study design could not determine the mechanisms underlying the risk and subsequent effect of PCP.Risk of PCP is aggravated in the kidney transplant cases with male gender, positivity for CMV, and non-use of oral prophylactic antibiotics. PCP significantly increases both the risks of mortality and graft failure. Accordingly, robust prophylaxis may be needed to prevent PCP and subsequent graft failure. The present results will be the basis of the future clinical trials on the use of prophylaxis in kidney transplant recipients.Additional file 1:Table S1. Information on the patients who had graft loss after the occurrence of Pneumocystis jirovecii pneumonia. Table S2. Information on the patients who died after the diagnosis of Pneumocystis jirovecii pneumonia. (DOCX 20 kb)


Dual kidney transplantation (DKT) offers a way to extend the use of kidneys from expanded criteria donors (ECDs). Here, we compared the outcomes of DKT with those of single kidney transplantation from standard criteria donors (SCDs) and ECDs.In 2014, we began performing DKT using both kidneys from deceased donors greater than 70 years of age with one of two risk factors: serum creatinine (sCr) level over 3.0 mg/dl or eGFR under 30 ml/min. By 2017, we had performed 15 DKTs. We compared the outcomes of the 15 DKT recipients with those of 124 patients who received a kidney from an SCD and 80 patients who received a kidney from an ECD.p < 0.01, < 0.01, and 0.03, respectively). DKT recipients were also older and had a higher diabetes burden than recipients of kidneys from ECDs and SCDs . DKT recipients had a lower nadir sCr and shorter duration to nadir sCr than single ECD KT recipients .Compared with ECDs and SCDs, DKT donors were older, had a higher diabetes burden, and a higher sCr level (The survival rates of DKT grafts were compatible with those of single KT grafts. Therefore, DKT may be considered a suitable an option to expand the donor pool. The rapidly growing incidence of chronic kidney disease and limited supply of donor organs has led to an increase in the number of patients awaiting kidney transplantation (KT). To overcome this organ shortage, kidneys from expanded criteria donors (ECD) or suboptimal donors are now widely used for transplantation. Importantly, the reported outcomes for KTs from ECDs are not inferior to those for KTs from standard criteria donors (SCDs) –3. HowevHere, we describe a single center experience and outcomes of DKT using kidneys from extremely marginal donors with age over 70 and acute kidney injury.en-bloc kidney transplantation, and 16 recipients who underwent multi-organ transplantation. The remaining 219 recipients included in the study were divided into three groups according to donor status. Group 1 (n = 124) consisted of patients who received a kidney from an SCD; Group 2 (n = 80) consisted of patients who received a kidney from an ECD; Group 3 (n = 15) consisted of patients who underwent DKT.This study was a retrospective single center historical cohort study and analyzed Samsung Medical Center electronic medical record and kidney transplantation database. We screened the records of 242 recipients who underwent deceased donor kidney transplantation (DDKT) between January 2014 and November 2017 in Samsung Medical Center. We excluded three pediatric recipients, four recipients who underwent All data analyzed in the study were derived from our institution’s electronic medical records and kidney transplantation database. The institutional review board of Samsung Medical Center approved this study protocol (SMC 2018–03-035) and waived the requirement for written informed consent.ECD was defined based on United Network for Organ Sharing (UNOS) criteria. ECDs consisted of deceased donors (DDs) older than 60 years and DDs 50 to 59 years of age who met two of the following criteria: (1) history of hypertension, (2) cerebrovascular accident as a cause of brain death, and (3) final pre-procurement sCr level > 1.5 mg/dL . DKTs weUrine leakage, ureteral stricture, post-operative bleeding, renal artery stenosis, and lymphocele requiring drainage were included as surgical complications. Graft failure was defined as the need for either permanent dialysis or re-transplantation. Delayed graft function (DGF) was defined as needing dialysis during the first week post-transplantation. eGFR values were calculated by the MDRD study equation.For DKT, we used a midline incision and implanted the graft kidneys in the intraperitoneal space separately on both sides. Anastomoses to the iliac vessels were performed separately on each side. Ureteroneocystostomies were performed bilaterally after revascularization of both kidneys using the Lich-Gregoir technique, with a double J stent for each ureter.2, single dose on day 0) was given to all recipients with DSAs. In all cases, the induction agent was ultimately decided according to the physician’s preference.We used rabbit anti-thymocyte globulin  as an induction immunosuppressive agent for recipients with donor-specific anti-HLA antibodies (DSAs) or history of KT, as well as in single ECD KT and DKT cases. Otherwise, we used an interleukin-2 receptor antagonist induction  as the induction immunosuppressive agent. A single dose of anti-CD20 monoclonal antibody . The Generalized Estimating Equation (GEE) was applied to analyze repeated measurements for sCr and eGFR levels. Statistical analyses were performed using SAS version 9.4 .2, and 2.1 mg/dL, respectively which represented the highest values among groups . More donors of the ECD and DKT groups had diabetes or hypertension  and died from cerebrovascular disease (p = 0.03). The kidney donor profile index (KDPI) and kidney donor risk index (KDRI) were highest in the DKT group . Recipient characteristics are summarized in Table p < 0.01). More diabetic recipients underwent DKT (p < 0.01). Patients with a previous history of KT, panel reactive antigen (PRA) over 50%, or DSAs were more prevalent in the SCD group . Mean cold ischemic times were not different among groups. Mean dialysis duration before KT in the DKT group (4.8 years) was shorter than in the ECD group (6.1 years), although the difference was not statistically significant (p = 0.17).Donor characteristics are summarized in Table p = 0.41). In terms of nadir sCr level and time to nadir sCr, DKT was comparable to single SCD KT and superior to single ECD KT (p < 0.01 and 0.02). Post-transplant eGFR one year after KT was lowest in the ECD group (p < 0.01). At two and three years after KT, eGFRs were lowest in the DKT group (p < 0.01 and 0.01); however, the eGFR and sCr trends were not significantly different among groups . The post-operative complication rate after DKT (13.3%) was not higher than that after single ECD KT . There was one lymphocele and one ureteral leakage in the DKT group. The lymphocele was controlled after percutaneous drainage, while the ureteral leakage was controlled after double J stent and Foley catheter insertion, which were removed two and four weeks later, respectively.Clinical outcomes are summarized in Table p < 0.01 in all). Disadvantages from the donor factors were overcome by doubling the number of transplanted nephron in DKT. Even though the difference was not statistically significant (p = 0.41), the rate of DGF after DKT (20%) was lower than that of single ECD KT (33.8%). It can be explained by that DKT can supply sufficient number of nephron and, even if some fraction of nephrons were injured, enough number of nephrons is preserved to facilitate primary function.Outcomes of DKT in our study were not different from those of single KTs in terms of graft survival rate and graft function after KT despite a higher age, higher sCr level, greater burden of diabetes, and higher KDPI and KDRI scores in DKT donors  compared to donors younger than 70 (7.4%). Additionally the discard rate in donors between 60 and 70 years was 9.6%. In a clinical setting with insufficient support by pathologists specialized in kidney allograft histology, donor age is the most important factor in the decision of a clinician to discard a kidney graft. Therefore, to reduce the graft discard rate it is necessary to use kidneys from older donors. Numerous studies have reported successful outcomes after DKT from donors older than 70 , 16–20. Even though our results were comparable to the results of single ECD KT, there should be criteria for clinicians to decline a graft kidney. We discarded kidneys when they were grossly discolored or atrophied, or when the donor sCr level had slowly and consistently deteriorated for a period longer than 5 days. Nevertheless, we agree that a histological scoring system is more objective if pathological assessment of the donor kidney is available. Further efforts to establish pathology support systems for kidney graft evaluation are thus necessary.p < 0.01). In our study, the eGFR level one year after KT was higher in the DKT group than in the ECD group. However, we found that this situation reversed at two and three years after KT. Although the trend of eGFR change was not significantly different in these groups (p = 0.65), it seems reasonable and prudent to transplant kidneys with the potential to last longer into recipients who are expected to live longer. Moreover, the DKT group showed a lower rate of DGF, a lower level of nadir sCr, and a shorter duration to nadir sCr, all of which can lower recipient’s physical burden during the immediate post-operative period. These factors can be helpful, especially for older recipients with less physical reservoir. The duration of dialysis prior to KT was shorter in the DKT group (4.8 years) than in the ECD group (6.1 years), although the difference was not statistically significant (p = 0.17). Considering the age of older recipients and the importance of performing KT as soon as possible, DKT should be viewed as a reasonable solution. In addition, Rigotti et al. improved DKT outcomes for older recipients with a personalized immunosuppression strategy [Our center utilized an “old for old” strategy. As a result, the mean age of recipients in the DKT group (63.7 yrs) was significantly higher than in the ECD group (55.2 yrs., nt p = 0.. Consident p = 0.. ConsideThere were several limitations to the present study. The number of DKT cases was relatively small and the follow-up duration was too short to draw long-term results. Given these limitations, the ability to generalize our results may be limited. Despite these limitations, this study is valuable because of the successful utilization of kidneys from extremely marginal donors for DKT. To the best of our knowledge, this is the first report of DKT outcomes in an Asian population. Out results also provide evidence for strategies to improve the use of kidneys safely from marginal donors.DKT provides comparable graft survival and graft function to single ECD KT despite older donors with higher sCr levels, more diabetes, and higher KDPI and KDRI scores. DKT is a safe and feasible way to use kidney grafts from extremely marginal donors, thereby reducing the organ discard rate. Further efforts should focus on avoiding inappropriate use of unacceptable kidneys and improving DKT outcomes.


Renal denervation (RDN) reduces sympathetic tone and may alter the sympathetic-parasympathetic balance. The autonomic nervous system is partly a regulator of innate immunity via the cholinergic anti-inflammatory pathway (CAP) which inhibits inflammation via the vagus nerve. Placental Growth Factor (PlGF) influences a neuro-immunological pathway in the spleen which may contribute to hypertension. The aim of this study was to investigate if modulation of renal sympathetic nerve activity affects CAP in terms of cytokine release as well as levels of PlGF.Ten patients treated with RDN (Medtronic Inc), were analyzed for TNF, IL-1b and IL-10 and Lipopolysaccharide (LPS)-stimulated cytokine release before RDN, 1 day after and at 3- and 6-months follow-up. Four patients who underwent elective coronary angiography served as disease controls (DC).p = 0.03). LPS-stimulated  TNF and IL-1b were significantly lower 1 day after RDN . IL-10 was significantly higher one day after RDN . These differences however declined during follow up. A more marked TNF reduction was achieved with a cholinergic analogue, GTS-21, in LPS-stimulated whole blood as compared with samples without GTS-21. Cytokine levels in controls did not differ before and 1 day after coronary angiography. PlGF was significantly higher in RDN patients and DC compared with healthy controls but did not change during follow-up.Baseline TNF was significantly lower 1 day after RDN  contains supplementary material, which is available to authorized users. Tissue injury, infection or ischemia triggers an immune response that generates signals in the sensory afferent vagus nerve. After reaching the central nervous system an activating response is subsequently returned via the efferent motoric part of the vagus nerve, referred to as the inflammatory reflex Tracey, . The sigHypertension is one of the most common non-communicable diseases with a high risk for long-term complications such as cardiovascular disease, stroke, and chronic kidney disease  > 160 mmHg) despite the use of 3 antihypertensive agents including a diuretic prescribed in optimal doses. Modulation of renal sympathetic nerve activity through catheter-based radiofrequency ablation (i.e RDN) has been introduced as an invasive add-on treatment for resistant hypertension, and has been evaluated in two European clinical studies (Symplicity 1/Symplicity 2)  reflects sympathetic and parasympathetic activity. HRV quantifies interval changes between hearts beat cycles. HRV is higher in healthy individuals than in patients with autonomic dysfunction. Reduced HRV is associated with adverse outcomes in hypertension, systemic inflammation, depression and is also associated with an increased risk for sudden death  or computer tomography-based (CT) angiography for mapping of renal artery anatomy. The RDN procedures took place between February and November 2013. Patients with any secondary cause of hypertension were excluded.Four patients scheduled for elective coronary angiography due to ischemic heart disease served as disease controls (DC). A summary of clinical characteristics of the RDN patients and DC is presented in Table RDN patients received pre-treatment with diazepam and peri-procedural analgesia with an i.v. infusion of remifentanil. Antithrombotic treatment included Aspirin  and a peri-procedure administration of a weight-adjusted dose (50–100 U/kg) of unfractionated Heparin (UFH) with a target activated clotting time (ACT) of 250 s.After routine preparation for catheterization, femoral artery access was established using standard 8 French (F) sheaths and guide catheters were advanced to selective left and right renal artery position. Radiofrequency ablation was performed bilaterally at a minimum of 6 locations in each renal artery using the EnligHTN™ multi-electrode renal denervation catheter . In the presence of multiple renal artery anatomy, all branches with a diameter of > 4 mm were treated. For post-procedure hemostasis, an arterial closure device  and provisional manual compression was used. Following the procedure, patients were monitored in hospital for 24 h with pre-discharge evaluation of BP, arterial access site hemostasis and renal function.Coronary angiography was performed in a standard fashion, predominantly through right radial artery access with standard 6F sheaths and catheters. All patients received pretreatment with Aspirin , paracetamol and diazepam. In the presence of coronary artery disease, a clinical decision on revascularization strategy was taken, and in case of ad hoc percutaneous coronary intervention (PCI), standard procedure included implantation of drug eluting stents (DES) and the addition of antithrombotic treatment with a weight adjusted dose of low-molecular-weight-heparin (LMWH) as well as a second oral anti-platelet drug . Post-procedure monitoring of BP, hemostasis and renal function were similar to that in RDN patients.All RDN patients and DC were sampled in the morning before and 1 day after intervention. Blood samples were analyzed for high sensitivity C-reactive protein (hsCRP), hemoglobin (Hb), White Blood Cell count (WBC) with differentials. RDN patients were sampled again at 3 and 6 months. HsCRP, Hb and WBC were analyzed by using routine methods at the Department of Clinical Chemistry either at Södersjukhuset or at Karolinska University Hospital.Escherichia coli 0111: B4, Sigma-Aldrich, St Louise, MO, USA, cat. No. L4130) solved in 1 mL PBS, sonicated for 30 min, vortexed well and diluted 5 times to 1 × 106 ng/mL. This solution was then serially diluted with PBS to LPS-concentration 10 and 100 ng/mL. A selective a7nAChR cholinergic agonist, GTS-21, was manufactured and shared by Yousef Al-Abed at the Feinstein Institute for Medical Research, Manhasset. New York, USA. Whole blood aliguots of 1 mL were incubated during 4 h with the LPS concentration of 0, 10 and 100 ng/mL and GTS 21 90 μmol/L.The whole blood assay, a standard method to test cholinergic function, was performed by a method previously described  and frozen at − 80 °C pending cytokine analyses. Plasma for baseline cytokine levels was obtained from the whole blood samples in heparinized tubes prior to stimulation and incubation.High sensitivity TNF, IL-1b and IL-10 were analyzed by immunometric assays on an Immulite 1000 Analyzer  according to the instructions of the manufacturer. according to the instructions of the manufacturer.High sensitive PlGF (hsPlGF) was analyzed at all timepoints in RDN patients and DC. Baseline levels were compared with a cohort of fifteen healthy individuals from two previously published studies; one comparing cytokine release between hemodialysis patients and healthy controls  for 20 min at sampling frequency of 300 Hz. Analysis of ECG signals was performed using MATLAB 2017b,  scripts. Pre-processing was done in order to remove the powerline interference, reduce baseline wanders and high frequency noises by using 60 Hz notch, 5 Hz high-pass and 35 Hz low-pass filters, respectively. R-Peaks were detected by Pan-Tompkins method  for ordinal /nominal variables. Comparisons between more than two groups were assessed with non-parametric ANOVA Kruskal-Wallis test for continuous variables. We performed no adjustments for multiple comparisons i.e. Bonferroni procedures. In the present study all p-values are descriptive as we were studying exploratory hypotheses. Statistical analyses were performed using statistical software SAS version 9.4 .All continuous data were expressed as median (10th to 90th percentile) and ordinal and nominal data as percentage. Statistical significance was set at the level of Baseline values of cytokines in RDN patients and DC are shown in Table p = 0.03) 1 day after RDN whereas hsCRP, Il-1b and IL-10 were not . Anti-inflammatory IL-10 levels were significantly higher on 1 day after RDN . However, at 3 and 6 months after RDN the LPS-stimulated cytokine levels were similar to levels before RDN in the incubated 4 h samples  of TNF-levels 1 day after RDN, that waned during follow-up  did not show any significant changes in RDN patients at 1 day or at 3 and 6 months after RDN  and (5-95), percentiles . Short-term norms: SDNN mean 50 (SD 16), (range 785-1169); RMSSD mean 42 (SD 15), (range 19-75); LF mean 519 (SD 291), (range 193-1009); HF 657 (SD 777), (range 83-3630); LF/HF; 2.8 (SD 2.6), range 1.1-11.6) (20). Still it has been shown that monocytes are a major source of TNF in the whole blood LPS model , and TNF  after RDN were apparent compared to baseline.  and 6 months (P < 0.01) after the procedure. Both our results and the ones by Zaldivia, utilizing different methods of monocyte activation, however, suggest that the effect may not be long-lasting, or possibly weaker with time.Although intervening in renal sympathetic-nerve activity may influence the cytokine release as shown in the whole blood model, very little is known about RDN and anti-inflammatory effects and the time-line of these potential changes. In a study by Zaldivia et al. a reduction of several circulating inflammatory markers including IL-1 Additional file 2:Table S2. HRV variables SDNN, SDMRR, HR, HF, LF/HF in RDN patients at baseline, day 1 and at 3 and 6 months. (XLSX 9 kb)