P = 0.002, n = 25). Plasma and kidney renin content determined by radioimmunoassay were significantly lower in LPK vs. Lewis . These parameters were not affected by RDN. Intrarenal mRNA expression levels of renin, angiotensinogen, angiotensin-converting enzyme (ACE)2, and angiotensin II receptor type 1a were significantly lower, whereas ACE1 expression was significantly higher in the LPK vs. Lewis . This pattern of intrarenal RAS expression was not changed by RDN. In conclusion, RDN does not affect hypertension or the RAS in the LPK model and indicates RDN might not be a suitable antihypertensive strategy for individuals with juvenile-onset PKD.We examined the effect of total and afferent renal denervation (RDN) on hypertension and the renin-angiotensin system (RAS) in a rodent model of juvenile-onset polycystic kidney disease (PKD). Lewis Polycystic Kidney (LPK) and control rats received total, afferent or sham RDN by periaxonal application of phenol, capsaicin or normal saline, respectively, and were monitored for 4-weeks. Afferent RDN did not affect systolic blood pressure (SBP) determined by radiotelemetry in either strain (n = 19) while total RDN significantly reduced SBP in Lewis rats 4-weeks post-denervation (total vs. sham, 122 ± 1 vs. 130 ± 2 mmHg,  Before progressing to end-stage renal disease, PKD patients commonly present with cardiovascular complications including hypertension, ischaemic heart disease and heart failure2. Excessive sympathetic nerve activity is believed to play an important role in the development of these cardiovascular complications3. Accumulating evidence also suggests the involvement of the renin-angiotensin system (RAS), both systemic and intrarenal, in the pathogenesis of PKD5. Intrarenal RAS refers to the local autocrine/paracrine system in the tissue, in this case the kidney, with all the components necessary to generate angiotensin II (Ang II) in situ6. Dysregulation of intrarenal RAS is described in human PKD, as evidenced by ectopic expression of renin in some cysts and dilated tubules9. Upregulation of intrarenal RAS components including renin, angiotensin-converting enzyme (ACE) and Ang II has been described in a rodent model of autosomal recessive PKD (ARPKD)10 (in the absence of any changes in systemic Ang) and similar findings were seen in mouse models with loss of cilia or polycystin 1, where cyst formation and increased blood pressure were associated with increased kidney angiotensinogen (AGT) levels11.Polycystic kidney disease (PKD) is a leading cause of end-stage renal disease, accounting for approximately 8% of patients receiving dialysis or renal transplantation in the United States12. There is also evidence they can impact intrarenal RAS, with renal denervation (RDN) reducing intrarenal renin protein and mRNA levels in a mouse model of neurogenic hypertension13. RDN also reduced blood pressure in these models.Complex interactions between the sympathetic nervous system and RAS activity exist in the kidney. Sympathetic nerves promote juxtaglomerular renin release in response to decreased blood pressure, driving systemic levels of Ang II14 and clinical studies15 has shown RDN can produce beneficial effects on blood pressure, cardiac and renal function. It is proposed that both sympathetic and sensory nerves are involved, as bilateral nephrectomy in chronic kidney disease patients reduces blood pressure and sympathetic activation, with the latter appearing to be mediated by an afferent signal arising in the diseased kidneys16. PKD would therefore seem to be an ideal candidate for RDN due to the presence of sympathetic activation and altered intrarenal RAS expression10. To date however, randomized clinical trials examining the impact of RDN in patients with PKD is lacking17, with only a limited number of case reports available, that do however, report a blood pressure lowering response19.In chronic kidney disease, data from preclinical20, whereas removal of afferent nerves is responsible for the response in the DOCA-salt hypertension model21.Both clinical and experimental RDN procedures typically destroy the renal sympathetic efferent and sensory afferent nerves; however, it is unclear whether the responses seen are due to the removal of sympathetic or afferent components, or both. The relative contribution may differ depending on the underlying disease aetiology. For e.g., the antihypertensive effect of RDN in the Dahl salt-sensitive model has been linked to the removal of sympathetic nerves22. The model presents phenotypically similar to ARPKD, developing early-onset hypertension, alongside a progressive decline in renal and autonomic function24, and increased renal sympathetic nerve activity26. We have shown that systemic RAS is reduced in this model23, however, to date, the intrarenal RAS has not been studied.The Lewis Polycystic Kidney (LPK) rat is a model of PKD arising from a mutation in Nek8, consistent with nephronophthisis (NPHP)9 in humansIn the present study we therefore tested the hypothesis that intrarenal RAS is upregulated in the LPK when compared to control rats, and that amelioration of this, through RDN, would limit the development of hypertension, cardiac autonomic and renal dysfunction. We undertook total RDN and selective sensory RDN as means to delineate the contribution of each component of the renal innervation to any outcomes observed.27.In Study 1, we firstly confirmed using immunohistochemistry that both total and sensory RDN, performed in 6-week-old Lewis and LPK animals, could ablate the relevant nerve populations, examining the kidney tissue one week after surgery. Representative immunohistochemistry images of perivascular tyrosine hydroxylase (TH) and pelvic calcitonin gene related peptide (CGRP) labelling as markers of sympathetic and sensory innervation, respectively, from Lewis and LPK rats one week after the different denervation protocols  and quantitative analysis are provided in Fig. P < 0.0001 between strains, F = 2.6, P = 0.10 between treatment groups, n = 28). Heart weight/body weight ratio was overall higher in LPK rats compared with Lewis controls but was unaffected by RDN procedures . This pattern was also observed for plasma urea . No overall strain or treatment effect was observed for plasma creatinine .We also assessed the impact of the RDN procedures on morphological and biochemical parameters in the above mentioned animals. One week after surgery, two-way ANOVA indicated that the kidney weight/body weight ratio was overall higher in the LPK compared with Lewis and that RDN had no effect  ratio was assessed in 3 Lewis animals and ≥ 3 LPK animals per treatment group. Urinary protein was only detected in 4 of 9 Lewis rats tested (range 0.09 to 0.32 g/L) and 5 of 12 LPK animals tested (range 0.07 to 0.58 g/L). Thus, insufficient numbers from each treatment cohort were available for a statistical comparison of creatinine normalised values between treatment groups.P < 0.05) was noted, with Lewis females having a higher SBP compared to male Lewis, and LPK females having a lower SBP and a higher HR compared with male LPK rats , diastolic blood pressure (DBP) and heart rate (HR). Overall, a sex effect in Lewis SBP and in LPK SBP and HR . In the Lewis control group, SBP and DBP increased slightly with age , and DBP  in Lewis, with post-hoc analysis revealing that animals which had received total, but not afferent, RDN had a lower SBP and DBP at 10 weeks of age compared to sham animals . In both strains, an age-related reduction in HR was observed .Consistent with the development of hypertension in the LPK reported previouslyP < 0.05), whereas in the LPK night-time and daytime SBP and DBP values were not significantly different . In both Lewis and LPK, night-time HR was significantly higher than daytime values . The effect of RDN on the diurnal variation of cardiovascular parameters, i.e., difference between night-time and daytime SBP (ΔSBP), DBP (ΔDBP) and HR (ΔHR) are presented in Supplementary Fig. P > 0.05).Diurnal variation in cardiovascular parameters for all Lewis and LPK animals is presented in Supplementary Fig. P < 0.0001), with total RDN producing a beneficial effect by lowering LF SBPV compared with sham RDN, evident at both 7 and 10 weeks old (P < 0.05). No treatment effect was noted for LF SBPV in LPK . There was also no effect on heart rate variability (HRV) .Cardiovascular autonomic function was assessed using power spectral analysis. In the Lewis, an overall treatment effect was noted in low frequency (LF) systolic blood pressure variability (SBPV)  Fig. C,D or baRV) Fig. E,F in LeP < 0.0001) and RDN had no effect on this  , renin, angiotensin-converting enzyme 2 (ACE2) and angiotensin type 1a receptor (Agtr1a) were lower and ACE1 expression greater in the LPK compared with Lewis (see Table P < 0.01) as well a lower creatinine clearance rate , confirming impairment of renal function and cardiac hypertrophy at this age. Urine protein was below the level of detection in 4 of 13 Lewis rats tested meaning insufficient numbers from each treatment cohort were available for a statistical comparison of UPC between treatment groups however UPC was higher overall in the LPK (both P < 0.001), confirming filtration barrier injury in the LPK. Urinary sodium measured over 24 h was not significantly different between LPK and Lewis . There was no treatment effect of RDN in any of these parameters in either strain  are shown in Fig. P = 0.13 and F = 2.3, P = 0.15 respectively), indicating reinnervation was well established. After afferent RDN in the Lewis at 10 weeks, there was no significant difference from sham animals for TH , as anticipated, but also for CGRP levels , indicating significant sensory reinnervation had occurred after this procedure. In the LPK, after total RDN, there was no significant difference between sham and denervated animals for TH at age 10 weeks . However, CGRP levels were significantly less . After afferent RDN, there was no significant difference from sham animals for TH , again as anticipated, but also for CGRP levels , indicating significant sensory reinnervation having occurred as seen in the Lewis.Based on studies that show sympathetic and afferent renal reinnervation occurs in the rat after renal denervationThe major findings of our study are that neither total nor afferent RDN affects blood pressure, autonomic or renal function in LPK animals and that total RDN, but not selective afferent ablation, produces a modest blood pressure reduction in Lewis control animals. In addition, intrarenal renin content, as well as mRNA for intrarenal RAS components, were significantly less in LPK vs. Lewis control animals, and this was not altered within either strain by either total or afferent denervation procedures. Lastly, anatomical evidence of regrowth of the renal efferent and afferent nerves was present at 4 weeks post total-RDN as detected by immunohistochemistry.30 and consistent with this, in the present study total RDN produced a modest reduction in systolic and diastolic blood pressure in the Lewis that was of comparable magnitude to that observed in other normotensive animal models after RDN31. We also observed an overall reduction in LF SBPV in the Lewis controls that received total RDN, evident within one week of the denervation, before the reduction of blood pressure. As the LF component of SBPV is contributed to by sympathetic modulation in rats32, a reduction in LF SBPV indicates a decrease in the sympathetic branch of autonomic cardiovascular control32, and suggests that a reduction in vasomotor tone underlies the blood pressure-lowering effect of total RDN.Renal sympathetic nerves play a tonic role in the regulation of blood pressure under normotensive conditions30. However, total RDN did not alter urinary sodium excretion, plasma or kidney renin content or gene expression levels of intrarenal RAS components in the Lewis strain. This is consistent with a previous study in which total RDN reduced blood pressure without affecting plasma renin activity in normotensive Sprague–Dawley rats33. It is possible that any initial response to RDN resulting in differences in sodium excretion relative to sham animals was corrected by pressure-natriuresis mechanisms by the time the 24 h urine samples were collected at age 10 weeks. It is also important to note that due to technical limitations, we measured 24 h urinary sodium excretion instead of the fractional excretion of sodium, which is the standard method for sodium excretion determination34. Further, we did not monitor 24 h sodium intake and output. Nevertheless, these findings do suggest that alterations in sodium excretion or intrarenal RAS are not a primary driver of the sustained decrease in blood pressure produced by total RDN in control rats.We further explored the mechanism by which total RDN reduced blood pressure in the Lewis animals by investigating if there were any changes in urine sodium excretion, plasma renin content or intrarenal components of the RAS following total RDN, all being important regulators of blood pressure35. There are a number of possible explanations for our contrasting observations. Firstly, unlike the Han: SPRD-Cy/ + PKD model, which presents with mild hypertension, the LPK model displays marked hypertension24 alongside elevated renal sympathetic nerve activity at a young age36. Noting the early onset of hypertension in these animals, it is conceivable that the timing of the RDN intervention in our study was not early enough to limit the effect of increased renal sympathetic nerve activity on the development or progression of hypertension in the LPK. Secondly, a positive association between blood pressure and renal size has been described in children, adolescents and adults with PKD38. Kidney volume was reduced in the Han: SPRD-Cy/ + model after total RDN35, but likely remained unchanged after RDN in our model as evidenced by no difference in kidney weight to body weight ratios when compared to the sham procedure. Notably, in the human studies the relationship between blood pressure and kidney size was found to be independent of the degree of renal dysfunction. In our study, neither total nor afferent RDN improved renal function. Within the context of PKD, the ability of RDN to limit disease progression is therefore likely dependent on the specific disease phenotype, including age of onset and cystic disease severity.A key finding of our study was that neither total or sensory RDN affected blood pressure or heart weight ratio in the LPK model of juvenile-onset PKD. This is in contrast to the only other published study examining renal denervation in a PKD animal model, where using Han: SPRD-Cy/ + rats, total RDN was shown to reduce blood pressure24. Unlike in the spontaneously hypertensive rat, where RDN has been shown to significantly improve cardiac and sympathetic baroreflex sensitivity independent of a blood pressure response40, neither total nor afferent RDN affected HRV, SBPV or BRS in the LPK. This suggests that in this model, other pathways drive the perturbed autonomic dysfunction that is evident.The LPK model of PKD exhibits marked autonomic dysfunction including increased cardiac sympathetic and reduced cardiac vagal regulation, and a reduction in cardiac baroreflex control that contribute to an overall increase in SBPV and reduction in HRV5. However, in the current study, we demonstrated reduced systemic renin content, agreeing with our previous studies that demonstrated reduced plasma renin activity in this model23, and, in contrast to our hypothesis, we demonstrated lower levels of kidney renin content and intrarenal expression of the RAS component genes (with the exception of ACE1 mRNA) compared to our control animals. This is in contrast to the pck rat model of ARPKD10, where increased mRNA expression of renin and Ang II staining in the kidney was observed, and human autosomal dominant PKD8 and ARPKD9, where ectopic expression of renin in cysts was observed. The mechanism responsible for the suppressed systemic renin in our model may well be a consequence of the severe hypertension (~ 230 mmHg vs ~ 150 mmHg in the pck rat at a similar age), given that pressure dependent feedback on juxtaglomerular renin secretion through this classical pathway is linked to increased systemic and renal reperfusion pressures41. The mechanism responsible for reduced intrarenal RAS is not clear from the results of our study, noting further, and again contrary to our hypothesis, total RDN did not have any significant effect on any of the components we assessed. This may be because any effect of RDN on intrarenal RAS was counteracted by other pathways involved in intrarenal RAS regulation such as the prorenin receptor, Wnt/β-catenin signaling, prostaglandins, Klotho and nuclear receptors (as reviewed by6). This is an area warranting further study, noting that in this model, despite low RAS levels, both systemically and as we have now established, intrarenal, RAS inhibition is an effective blood pressure lowering strategy44.Activation of the RAS, both systemic and intrarenal, have been proposed as mechanisms for hypertension development in PKD45 and as such, changes in our indicators of intrarenal RAS in response to RDN may have been masked. In the literature, the impact of RDN on both systemic and intrarenal RAS has shown to be variable. Following RDN, a decrease in plasma renin activity has been shown in a rabbit model of hypertension secondary to chronic kidney disease14 and a decrease in renal renin is seen in a mouse model of neurogenic hypertension13, while in the spontaneously hypertensive rat model of essential hypertension, total RDN had no effect plasma renin activity or kidney renin content46. In human clinical studies, RDN similarily had no impact on circulating renin in patients with resistant hypertension47. This suggests that the variable effects of RDN on RAS, both systemic and intrarenal, are mediated by the underlying mechanism in the different disease states.An important consideration in the interpretation of this data is that the collection of blood samples was taken in animals after the administration of anaesthetic. Anaesthesia is known to activate the intrarenal RAS29. While direct evidence of nerve regrowth after RDN in humans is lacking, renal sympathetic nerve regrowth is observed at 5 months post kidney transplantation48 and in a sheep model of catheter-based renal denervation, 11 months post procedure there is both functional and anatomical evidence of reinnervation49. What is unclear is whether renal reinnervation following RDN has any impact on the observed blood pressure response. In the present study, despite anatomical evidence of renal sympathetic and sensory nerves 4 weeks post procedure, the blood pressure of Lewis rats that underwent total RDN was still significantly lower than sham animals. If renal reinnervation does not dampen the initial blood pressure lowering effect, it would explain why a sustained response to catheter-based RDN lasting 3 years after the initial procedure has been observed in clinical studies50. This is also consistent with the spontaneously hypertensive rat model, where a second RDN procedure does not further reduce blood pressure51. An important caveat when interpreting our results, however, is while results using immunohistochemistry are qualitatively quite clear, due to the cystic features of the LPK kidneys, we were unable to apply unbiased stereology methods using random sections from the whole kidney. Our must therefore be considered as a semi-quantitative measure and cannot be directly translated into a complete loss of either innervation or functionality. Additional measures such as tissue levels of CGRP or noradrenaline, and functional validation of the denervation, for example, as undertaken in the study by Booth et al. in a sheep model of RDN49 would add to the strength of evidence. Another limitation of these studies is the sample numbers for both these groups (Studies 1 and 2b), which are relatively small. A posthoc power analysis indicated that the study power is over 80% for each one however which would indicate additional animals would be unlikely to alter the result.A final aspect of our study was evidence of reinnervation in both Lewis and LPK rats after the RDN procedures. This is consistent with reports of both functional and anatomical reinnervation of the rat kidney by 4 weeks post-RDN52. It is crucial to determine which patient populations will benefit from the approach and, just as importantly, those which will not respond, thereby preventing unnecessary invasive procedures. Our study has demonstrated that neither total nor afferent RDN has a positive effect on blood pressure, autonomic function or renal function in a juvenile onset rodent model of PKD, suggesting that this procedure would not benefit patients suffering from comparable juvenile onset ARPKD or NPHP. Alternative therapeutic approaches to limit hypertension and renal dysfunction will be required.The clinical implications of our work should be considered in context of the conflicting results surrounding clinical trials using catheter-based RDN studiesad libitum. At the end of the study, animals were euthanised with an i.p. injection of 20% (v/v) solution of sodium pentobarbital  prior to the collection of blood and tissues.All experiments were approved by the Animal Ethics Committee of Macquarie University and conducted in accordance with the Australian code for the care and use of animals for scientific purposes (8th Edition). The study was conducted and reported according to the ARRIVE guidelines. Lewis and LPK rats of both sexes  were obtained from the Animal Resource Centre, Perth, Australia and housed at the Central Animal Facility of Macquarie University on a 12-h light–dark cycle (lights on 6 am), at 22 ± 2 °C with access to food and water  29; (ii) afferent RDN by periaxonal application of 33 mM capsaicin29; or (iii) sham RDN by periaxonal application of normal saline. In Study 1, animals were euthanised one-week post-denervation or sham procedure (age 7 weeks), and kidneys collected for immunohistochemical assessment of renal sensory and sympathetic nerve innervation . In Study 2a, animals were instrumented with radiotelemetry probes and blood pressure and HR monitored to 10 weeks of age . In Study 2b, some of the animals (n = 9) from Study 2a and an additional group of animals were euthanised at age 10 weeks and biological samples including urine, plasma, heart and kidneys collected for subsequent analyses , including immunohistochemistry to assess what degree renal sensory and sympathetic nerve re-innervation had occurred at the end of the study period. A full breakdown of total animal numbers and sex used in each Study is provided in Supplementary Table The experimental protocol and study groups are illustrated in Supplementary Fig. 2 and animals were maintained on 2–3% isoflurane in 100% O2. Pre-operative analgaesia  and antibiotics  were administered.For all surgical procedures, anaesthesia was induced with 5% isoflurane in 100% O, the renal arteries were stripped of all visible nerve bundles and painted with 10% phenol in absolute ethanol; for selective afferent RDN the renal artery was painted with 33 mM capsaicin  dissolved in 5% ethanol and 5% Tween 80 in normal saline 2–3 times at 2–3 min intervals. Sham surgery entailed visualisation of the renal artery and painting with normal saline. Animals were provided with postoperative analgaesia  and supplementary fluids were administered once daily for 24–48 h postoperatively if required.Bilateral midline flank incisions were made to expose the right and left renal artery and vein. For total RDNAt the time of the RDN procedure, animals in Study 2a had radiotelemetry probes  implanted into the aorta via the right femoral artery with subcutaneous device placement. Animals were allowed to recover for 7–10 days before any radiotelemetry recordings were made.Arterial pressure (AP) was continuously recorded for 5 min every 15 min for 48 h once weekly from 7 to 10 weeks of age . From the AP waveform, SBP, DBP and HR were derived offline using Dataquest ART  and exported as text into Microsoft Excel . All recordings over the 48 h period were averaged to create a mean 24 h value for analysis. For assessment of diurnal variation, all recordings during the daytime (6 am to 6 pm) or night-time (6 pm to 6 am) were averaged to create a mean daytime or night-time value of cardiovascular parameters.24. A single average was created from all the 80 s segments per age point studied for the final analysis.To assess HRV, SBPV and BRS, which are surrogate markers of cardiovascular autonomic function, all AP waveforms acquired during the 48 h recording were imported into Spike 2  and the segments between 11 am and 1 pm (day) and 11 pm and 1 am (night) were chosen for subsequent analysis. The pulse interval was derived from the AP waveform and uniformly resampled at 10 Hz. 80 s segments of AP that were stable and where no ectopic beats were present were selected for analysis. A minimum of 6 segments consisting of 3 during the day and 3 during the night periods were identified for analysis for each animal at each age point sampled. Power spectrums for each 80 s period were generated using a fast Fourier transformation , exported as text into Microsoft Excel, then total power , very low frequency , low frequency , high frequency  power values of HRV were calculated. SBP was then derived from the same segments used to estimate HRV, uniformly resampled at 10 Hz and a power spectrum generated  with TP (0–3 Hz), VLF (0.04–0.2 Hz), LF (0.25–0.75 Hz) and HF (1–3 Hz) SBPV power calculated. Similarly, BRS was estimated on the same 80 s segments of AP as HRV and SBPV, using cross-spectral analysis of LF and HF HRV and SBPV power as previously describedRenal function was determined by measurement of plasma urea, plasma creatinine, and estimated CCR, as surrogate for glomerular filtration rate. As a measure of filtration barrier injury, urine protein was determined and normalised to urine creatinine level, expressed as urine protein to creatinine ratio (UPC). For urine collection, animals were acclimatised to metabolic cages, and then held for up to 5 h (from 9 am to 2 pm) to collect spot urine samples (Study 1) or for 24 h (from 9 to 9 am (Study 2a and 2b), during which time 24 h urine output and 24 h water intake were measured. Animals were provided free access to food and water in metabolic cages. Urine samples were spun and analysed immediately or frozen until analysis.At the termination of the study period, animals were euthanised as described above and blood (3–4 ml) was collected via cardiac puncture into ethylenediaminetetraacetic acid (EDTA) tubes . The samples were spun and the plasma was extracted, snap frozen in dry ice then stored at − 80 °C until further analysis. Plasma and urine samples were analysed using an IDEXX VetLab analyser . CCR was estimated using the following equation:2O). Data acquisition parameters of sample uptake time (20 s), stabilisation time (20 s) and read time (20 s) were used. All parameters, including sample dilution, were optimised in a series of preliminary experiments. Analysis consisted of washing the system with distilled water for 60 s and then running a series of procedural blanks, calibration sodium samples diluted to a range of 0.625 to 10 mg/l using sodium standards (Sigma-Aldrich) and the diluted urine samples sequentially. A repeat analysis of calibration samples was then performed to ensure the accuracy and precision of the procedure. For each sample, five readings were obtained and averaged to create a final concentration reading in mmol/l. Urine sodium concentrations were then normalised using 24 h urine volume and expressed as mmol/24 h.Urine sodium from animals in Study 2b (n = 13 Lewis and n = 12 LPK) were analysed using the 4100 MP-AES system , a microwave plasma atomic emission spectrometry-based technique, with urine samples diluted to 1:1000 (Lewis) and 1:500 (LPK) in distilled water (dH2O for subsequent incubation. Frozen kidney tissues were thawed and a representative sample of 0.2 to 0.5 g tissue obtained. This was homogenised in 5 mM EDTA Na2 (1/10 wt gram/vol mL) using a BIAOMA FJ-50 homogeniser/blender for 30 to 60 s at ½ speed at 0 °C. Homogenates were immediately centrifuged at 1000×g for 10 min, supernatant collected and left at 0 °C for 1 h and then frozen at -20 °C. The supernatant was diluted with dH2O or 0.9% saline 1/1000 and 25 µl  used for subsequent incubation.The renin content from plasma and kidney samples was determined by measuring the amount of Ang I generated per hour from added substrate using radioimmunoassay undertaken by ProSearch International Australia . For plasma, blood samples collected via cardiac puncture as detailed above were spun and 1–1.5 ml plasma extracted and stored at − 80 °C until analysis. A total of 2.5µL of plasma was diluted to 25 µl with dH2EDTA, 3 mM 2,3-dimercaptopropanol and 6 mM 8 hydroxy quinoline buffered with 150 mM sodium phosphate buffer pH 6.2. In preliminary experiments, varying volumes of sample were incubated for varying times to ensure that reaction kinetics were linear and not substrate limited. Subsequent incubations at 37 °C were for 1 h and were terminated by the addition of 925 µl dH2O and placing the tubes into boiling water for 4 min. The Ang I generated was measured by radioimmunoassay53 and converted to a rate of production known to be linear (zero order kinetics) and expressed as ng Ang I/ml/h (plasma) or ng Ang I/mg/h (tissue) at 37 °C. Intra assay variability  was 93 ± 10.2 ng/ml/h, 11%. Inter assay variability was − 91 ± 11.8, 13%. The IC 50 is at 0.7 ng/ml. Cross reactivity to angiotensin II and angiotensin (1–7) is less than 0.1%.The prepared plasma and kidney samples were then incubated at 37 °C with 25 µl of nephrectomised sheep plasma and 25 µl of an angiotensinase inhibitor cocktail containing 30 mM Nahttps://www.moma.dk/normfinder-software54), by identifying the pair with the most stable expression across the two animal strains in kidney tissue. Conditions for qPCR were as follows: 20 s at 95 °C, 20 s at 60 °C, and 15 s at 95 °C, 1 min at 60 °C, and 15 s at 95 °C for 40 cycles, then held at 4 °C. No reverse-transcriptase cDNA mix (− RT) and no template controls were also run on each plate to ensure experiment fidelity. Following qPCR cycling, the products were validated by melt curve analysis.For intra-renal gene expression analysis, total RNA was extracted from left kidney tissue (30 mg) using the SV total RNA Isolation System  and eluted in up to 50 µl of water and stored at − 80 °C. The RNA concentration of each sample was then determined with a Nanodrop 2000 spectrophotometer . The RNA (1 µg) was then reverse-transcribed with the AffinityScript QPCR cDNA synthesis kid  according to the manufacturer’s instructions. Quantitative-PCR was performed in 20 μl reaction volumes containing 1 μl of cDNA mix, Applied Biosystems PowerUp SYBR Green Master Mix  and gene primers (See Supplementary Table −(ΔΔCt ± Standard Error (SE) of the ΔCt) method was used55. To analyse a strain effect, data was presented as a relative expression of the gene of interest in Lewis animals being the reference group. To analyse a treatment effect within each strain, data was presented as a relative expression of the gene of interest with sham animals being the reference group.For relative gene expression level determination, the average Ct value of three replicates for each animal was normalised to the expression of two housekeeping genes β-actin and cytochrome C1. Values were then normalised to housekeeping genes . ΔCt values were used for subsequent statistical analysis. For determination of fold difference in gene expression between treatment groups within each strain or between two strains, the 22PO4/Na2HPO4, pH 7.4) at room temperature for 2 h, followed by 48 h incubation in the same blocking solution containing the primary antibody against CGRP or TH , and stored in 30% sucrose for cryoprotection. Then 14 µm coronal kidney sections were cut using a Leica CM1950 cryostat and mounted on superfrost plus slides . The sections were incubated in blocking solution containing either 20% normal rat serum , 20% donkey serum  (for CGRP immunohistochemistry) or 10% donkey serum (for TH immunohistochemistry), as well as 0.3% Triton-X 100 and 0.05% thimerosal  in Tris phosphate buffered saline  for kidney regions49 could not be applied. Instead, the level of TH staining was quantified in the immediate vicinity of blood vessels in the cortico-medullary junction (a region well established to be high in sympathetic innervation30), using a minimum of 4 arteries from each animal based on previous studies28. Innervation was measured no further than 30 µm from the vessels, incorporating both adventitial fibers and associated perivascular nerve bundles but excluding the vascular wall, and expressed as percentage of positive staining within perivascular area. Blind analysis was not possible as the main researcher (SL) performing the analysis was also aware of the group assignment. A minimum of 4 animals per treatment group per strain at each time point (one week and 4 weeks post RDN) were analysed.Because renal sensory nerves are almost exclusively located in the renal pelvisFollowing cryosection as described above, kidney sections were incubated for 2 h at room temperature in blocking solution containing 10% donkey serum in TPBSm as detailed above. Sections were then incubated with primary antibody for renin, in combination with one of the tubule regional markers Table  for 48 hSections were viewed using a ZENPRO epifluorescence microscope (Zeiss). Representative areas labelled with renin and/or tubule markers was imaged using either 10 × or 20 × objective depending on the size of the region of interest. A total of three animals from each strain were imaged.Data analysis was performed using GraphPad Prism  and IBM Statistical Package for the Social Sciences . Results are expressed as mean ± SEM when appropriate. Preliminary analysis of the data included testing for normality using an Anderson–Darling test.Immunohistochemical labeling between the three RDN groups within each strain was analysed using one-way ANOVA with Bonferroni’s post hoc test when needed. When comparing morphometric and biochemical parameters for both 7-week and 10-week old cohorts, two-way ANOVA was used with strain and treatment as the two variables. When comparing cardiovascular and autonomic function parameters, a two-way ANOVA was used with treatment and age as the independent variables within each strain. If either a treatment or age effect was noted, the data were further analysed using a Bonferroni’s post hoc test to determine group differences. If data was not normally distributed, it was analysed using a nonparametric Kruskal–Wallis test.For comparison of kidney and plasma renin content and gene expression analysis, a univariate general linear model analysis of variance was used to identify strain or treatment effects followed by Tukey post hoc analysis as indicated. If the data displayed unequal variance (Levene’s Test), Games-Howell post hoc analysis was used.Significance was defined as a P value < 0.05 for all analysis.Supplementary Information.


The Safeguarding the Brains of our smallest Children (SafeBoosC) project was initially established to test the patient-relevant benefits and harms of cerebral oximetry in extremely preterm infants in the setting of a randomized clinical trial. Extremely preterm infants constitute a small group of patients with a high risk of death or survival with brain injury and subsequent neurodevelopmental disability. Several cerebral oximeters are approved for clinical use, but the use of additional equipment may disturb and thereby possibly harm these vulnerable, immature patients. Thus, the mission statement of the consortium is “do not disturb—unless necessary.” There may also be more tangible risks such as skin breakdown, displacement of tubes and catheters due to more complicated nursing care, and mismanagement of cerebral oxygenation as a physiological variable. Other monitoring modalities have relevance for reducing the risk of hypoxic-ischemic brain injury occurring during acute illness and have found their place in routine clinical care without evidence from randomized clinical trials. In this manuscript, we discuss cerebral oximetry, pulse oximetry, non-invasive electric cardiometry, and invasive monitoring of blood pressure. We discuss the reliability of the measurements, the pathophysiological rationale behind the clinical use, the evidence of benefit and harms, and the costs. By examining similarities and differences, we aim to provide our perspective on the use or non-use of cerebral oximetry in newborn infants during intensive care. Care of patients during acute illness has improved dramatically during the last 70 years , and now routinely includes electronic continuous monitoring of vital functions and replacement therapy for organ dysfunction. It has typically been developed in a mechanistic, “plumber-like” manner, addressing one problem after the other within the “body-as-a-machine” paradigm. This is also true for newborn patients, although their small size, developing physiology as well as the fragility of their tissues pose special problems.The adding of elements to the “life-support” package has been done over the years with variable levels of evidence. Marketing of medical devices, such as electronic patient monitors, is possible with documentation of safety and compliance with “essential performance.” For comparison, pharmaceutical products can only be marketed with proven benefits to patients. A central element in the process is the randomized controlled trial (RCT).However, it takes an RCT with more than 1,000 patients to test if an intervention can reduce the risk of a complication from 10 to 5%. An even smaller reduction may be worthwhile, and it is hardly imaginable that all elements of life-support can be tested by RCTs. Each element can only be expected to contribute marginally to patient-relevant outcomes such as mortality, days alive outside hospital, motor or cognitive development, or the risk of neurodevelopmental disability.Diagnostic or monitoring methods can only affect outcomes, if they lead to changes in management. Thus, a trial that tests the effect of a diagnostic tool on patient-relevant outcomes must have “thresholds of action” and be coupled to potentially effective interventions. Furthermore, when testing the value of a diagnostic method, it is the “added value” that matters. Typically, measures of pathophysiology may “overlap” as guidance for interventions, e.g., an abnormal value in one variable such as blood pH makes it more likely that another variable is abnormal, such as blood pressure, capillary refill or skin color. In a clinical trial, the experimental diagnostic method is typically tested in addition to “management as usual.” Finally, the patient-to-staff ratio and staff experience may influence the added value.We will focus on four modes of continuous  electronic monitoring that all have as a purpose: To “safeguard the brain” by providing early warning of threats to its oxygen supply. Their clinical use differs markedly. Pulse oximetry is universally applied in neonatal intensive care and the use of invasive blood pressure monitoring is probably available in all neonatal intensive care units (NICUs) but used in an individualized way according to the precise clinical context. However, cerebral oximetry is used routinely in some NICUs and never in others, while electric cardiometry is not routinely used.Oxygen sufficiency of the brain depends on oxygen delivery, i.e., blood flow, blood oxygen content, and distances of diffusion from the capillaries to the mitochondria on one side, and oxygen consumption on the other. Blood flow is driven by pressure against vascular resistance, and blood hemoglobin concentration and the oxygen saturation of arterial blood determines the payload of oxygen. Blood pressure is generated by cardiac output against the total systemic vascular resistance.Loss of function  is the first consequence of acute oxygen insufficiency, and cellular injury occurs next. After an abrupt interruption of oxygen delivery, e.g., by cardiac arrest, injury starts within minutes, while it takes longer for injury to start when some oxygen is still delivered, e.g., in case of severe bradycardia, hypotension or hypoxia . The effHypoxic-ischemic brain injury is relatively common in newborn infants, and it is most well-defined after a complicated birth, postnatal collapse, or neonatal stroke. It is now generally accepted that hypocapnic ischemia is a cause of white matter disease and cerebral palsy in preterm infants . AlthougThe cardio-vascular transition after birth has specific aspects, including the potential for shunting through the arterial duct and the foramen ovale with increased pulmonary resistance. Filling of the heart may be compromised by positive airway pressure and the limited distensibility of the immature myocardium. The diving reflex is very active before and after birth, providing resilience during acute hypoxic stress to “vital” organs. While the brain is commonly counted as a vital organ, this is true for the brainstem, but may not be true for the hemispheres .A simple model of neonatal circulation from a clinical perspective is illustrated in Cerebral near-infrared spectroscopy has been used in clinical research since the late 1970s. The method used to provide absolute values of hemoglobin-oxygen saturation as a percentage from 0 to 100%, appeared in the late 1990s. Cerebral oximetry was rapidly applied in the context of cardiac surgery and is used almost universally as clinical routine in the perioperative care of newborn infants with congenital heart disease and to some degree, also more generally in neonatology . Its mecRandomized clinical trials have demonstrated that it is possible to reduce the “hypoxic burden” on the brain if the clinician has access to a real-time display of cerebral oxygenation when stabilizing very preterm infants in the delivery suite  or when Tissue oximetry reflects the saturation in the circulating blood within the blood vessels according to their volume. It is therefore “venous-weighted,” and thus sensitive to the balance between local oxygen delivery and oxygen consumption. Tissue oximetry is not dependent on pulsation, and in the approved devices for clinical use, it is assumed that the tissues beneath the optode are optically homogenous. Inadequacy of this assumption may be the main reason for the limited repeatability, i.e., the standard deviation of repeated measurement after placing and replacing the sensor on the head of a newborn infant is typically around 5%. Recently, it was demonstrated that even the reactivity to arterial desaturations may vary considerably after replacement of the optode .Only recently, an international standard has been issued  and due Comparison of tissue oximetry to jugular venous oxygen saturation in pediatric patients showed limits of agreement of −12.5 to +10.1%, which is better than in adults (−20.7 to +19.7%) . HoweverFinally, even end-capillary oxygen saturation does not accurately estimate mitochondrial oxygen tension due to variable diffusion distances, e.g., due to capillary loss and or tissue edema.The concept of pulse oximetry became clinically available in the early 1980s and became standard of care for anesthesia in The United States in 1986. Pulse oximetry is universally used in neonatology and depends on illumination of a significant arterial vascular bed and a measurable pulsatile signal. Co-oximetry on arterial blood provides a gold-standard for calibration and validation. Pulse oximeters approved for clinical use must have an accuracy (absolute root mean square) better than 3% in the range 70–100% as determined on healthy, adult volunteers. Unfortunately, accuracy may be less at oxygenation levels below 70%, and the agreement with co-oximetry was demonstrated to be surprisingly poor in a large study, using electronic patient data from clinical care in newborns .Electrical cardiometry was developed in the early 2000s but has not gained any significant role in clinical care, perhaps partly due to the increasing availability of neonatologist-performed echocardiography or targeted neonatal echocardiography, the term used in North America.Electrical cardiometry uses a weak high-frequency alternating current and four skin electrodes, to determine the pulsatile change in impedance or phase shift. The main signal source is assumed to come from the varying “alignment” of erythrocytes in the blood in the descending aorta, induced by the rapid changes in flow velocity—higher flow causes erythrocytes to align in columns. The signal is calibrated in the device and results are provided in common units as liters per minute and thus, can be directly compared to other measures. Limits of agreement when compared to echocardiography is in the range of ±30% . There iThe first descriptions of monitoring the blood pressure through the umbilical artery in the hours after birth, appeared in the late 1960s. Invasive monitoring of arterial blood pressure is an established part of newborn intensive care, although clinical care guidelines may only offer broad advice to its use .Arterial cannulas are also placed to ease blood sampling, partly for optimal blood gas measurement, and partly to reduce pain and stress when needing frequent blood sampling. Shortly after birth, umbilical artery catheters can be used. Recording blood pressure is technically simple and provided adequate zeroing, has few sources of errors. Blood pressure measurements derived from arterial catheters represent the gold-standard value, whereas the alternative, oscillometric, non-invasive measurement of blood pressure may not really be sufficiently reliable .Strong evidence of clinical benefit from randomized clinical trials is not available for any of the four modalities.For cerebral oximetry, an attempt is currently in progress to obtain such evidence by a pragmatic randomized clinical trial, the SafeboosC-III trial (NCT03770741), enrolling 1,600 extremely preterm infants, to test the hypothesis that the risk of death or survival with severe brain injury can be reduced from 34 to 26.5% . Also, tAlthough pulse oximetry has been standard of care during anesthesia since 1986, a Cochrane meta-analysis including five trials that enrolled 22,000 patients during perioperative care, concluded that the evidence of clinical benefit was very weak . In neonThe range of “normal” blood pressure has been widely discussed in the literature as the basis for treatment strategies. Ways to increase blood pressure have been widely studied in controlled trials. The clinical benefits, however, has mainly been studied using epidemiological methods, and only very recently a randomized, but unfortunately very underpowered, clinical trial was completed . As far The sensors used for cerebral and pulse oximetry may cause skin injuries due to a combination of heat and pressure. These sensors as well as the electrodes of electrical oximetry cause disturbance and may cause skin abrasions in the immature newborn, whereas the light and the electrical currents can be assumed to be without risks. The cannulas required for invasive blood pressure monitoring can cause thrombosis or embolism and result in loss of fingers, toes or part of extremities and in very rare cases, be life threatening.In principle, for all four modalities there are risks involved in change of management induced by faulty readings or by the limited understanding of the pathophysiology involved. For instance, thresholds for action are not well established.The costs of all four modalities are modest, at least when compared with the cost of some drug treatments, and when compared with the total cost of intensive care. Although there is an investment in monitors, and the sensors for cerebral oximetry are relatively expensive, the most important cost is probably the staff time involved in establishing and maintaining monitoring, as well as handling the results.There are probably more similarities than differences between the four modalities. One hindrance to more widespread clinical uptake of cerebral oximetry in neonatology, may be the complexity of the physiology, doubts about the “added value,” and the sensor costs. In contrast, the main motive for introducing pulse oximetry may have been to protect against excessive oxygen therapy and the risks of retinopathy, and presumed oxygen toxicity in other organ systems. Furthermore, the direct response to changes in the fraction of inspired oxygen makes pulse oximetry intuitively useful.output in situations with variable shunts through fetal channels, as well as the limited means of managing cardiac output. In some respects, monitoring of blood flow to relevant organs would be more appealing, but unfortunately, measuring cerebral blood flow with cerebral oximetry by NIRS appears to be even less precise than measuring tissue oxygenation (Invasive blood pressure monitoring has been readily available in all neonatal intensive care units for many years. Still, it may be used less often now because of the concern that blood pressure is not really relevant, but blood flow is. Clinical use of electric cardiometry providing a measure of flow, however, is likely to have been held back by the limited reliability in terms of agreement with echocardiography, by the reservations about the meaning of cardiac genation .On one side, as clinicians, we want to do the best for our patients. If a patient is critically ill and the balance of evidence is toward benefit, and our understanding is that it is mechanistically plausible that the patient should benefit, we may just use the technology to give them the best chance. This has been called compassionate use. We may end up implementing new interventions despite lack of robust statistical evidence. It may also provide comfort for parents and staff alike to know that “everything possible” has been done.On the other side, as health service providers, we strive to deliver rational care based on evidence provided by RCTs, realizing the rising costs of medical care as well as the risks of medicalization, overdiagnosis, and overtreatment.So, randomized clinical trials may have a place. The first question before planning a trial is if we have sufficient equipoise to inform parents and ask them for permission to let the child be randomized. It matters whether the trial will test something as a new option or something that has already been taken into routine use . TherefoThere have been some examples of widely used intensive care treatments that have been “rolled back” after randomized trials after an intervention had achieved widespread implementation. The hydroxyethyl starch for volume expansion is one such example after an RCT showed that it increased, not decreased, mortality . In neonThe challenge of providing strong evidence is increased when it is extracted by meta-analysis from several RCTs, since it can be argued that repeated meta-analysis as well as heterogeneity among trials should be accounted for, e.g., by trial sequential analysis. This is fair, since a given intensive care element is likely to become an element in different life-support care packages. Patient populations are also likely to differ among hospitals, regions and countries. However, trial sequential analysis increases the required information size further and may make apparently conclusive metanalyses inconclusive—also those who point in the direction of harm .The clinical uptake of the four monitoring modalities differs widely and this is not based on clear differences as regards mechanistic rationale, nor evidence of clinical benefits. Rather, a variety of reasons may be in play.Insufficient repeatability as well as unexplained differences among commercial devices is a limitation for cerebral oximetry. Only recently, an international standard has been published addressing the inability to extrapolate validation in healthy adult volunteers to sick patients or the anatomy of immature brains .The SafeBoosC-III randomized trial testing the benefits and harms of cerebral oximetry during the first days after birth in extremely preterm infants will hopefully be published mid-2022 and will inform the decision as regards its use as clinical routine in this small, but high-risk group of patients.Furthermore, it could be useful for other neonatal patient groups. The COSGOD-III trial tests the clinical benefit in the delivery suite. Preparations for a randomized trial in neonates in need of mechanical ventilation, SafeBoosC-IIIv are being made, with the goal of enrolling 3,000 newborns. The aim is to test the hypothesis that the risk of death or survival with moderate or severe neurodevelopmental disability at 2 years of age can be reduced from 20 to 16% by the addition of cerebral oximetry to the “intensive care package” that these children need. We invite others to join this effort .More randomized trials will be useful, but questions will remain regardless of the results of these trials. Do the trials represent the conditions in my neonatal unit? If the trials are “negative,” are relevant effects really excluded?Therefore, a better understanding of the causal pathways , better The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.GG drafted the first version. All authors discussed the substance, critically reviewed the draft, made changes, and approved the final version.Elsass foundation is the main sponsor of the SafeBoosC-III trial from where this perspective.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Exercise is recommended as an effective lifestyle behaviour for adults to prevent and treat hypertension. In this study, a randomized-effect meta-analysis was used to analyse the influence of exercise interventions on blood pressure in patients with hypertension.  Candidate papers were retrieved from PubMed, Web of Science, Embase, and Cochrane Library electronic databases, and 46 studies were finally included and analysed.  It was shown that preplanned walking (systolic blood pressure (SBP): WMD (weighted mean difference) = −5.94, 95% CI: −8.57, −3.30; diastolic blood pressure (DBP): WMD = −2.66, 95% CI: −3.66, −1.67), yoga , aquatic sports , and football  had significant effects on blood pressure reduction. However, Tai Chi  and Qigong  did not significantly reduce blood pressure. The heterogeneity of the meta-analysis was high.  Walking, yoga, aquatic sports, and football were feasible and independent lifestyle interventions, and they were effective options for treating hypertension. More scientifically designed randomized controlled trials are needed in the future to further compare different forms of exercise for the treatment of hypertension. With the rapid development of the social economy and the improvement of living standards, the prevalence of cardiovascular diseases is increasing. Hypertension is the most important risk factor for cardiovascular disease and should be given global attention. The World Health Organization (WHO) estimated that 25% of adults worldwide have hypertension, accounting for 4.5% of the global disease burden . The numRuptured blood vessels caused by hypertension could be life-threatening . The magExercise is a planned, structured, and repetitive activity carried out in leisure time to improve or maintain physical function. According to the WHO recommendations and guidelines for the diagnosis and treatment of hypertension in China (2019), increased aerobic exercise has been used as a supplement to antihypertensive drugs . EvidencSeveral cohort studies, randomized controlled trials (RCTs), and meta-analyses have explored the effects of aerobic or endurance exercise on blood pressure in patients with hypertension –16. Howe“Hypertension” OR “Hypertensive” OR “High blood pressure” OR “Mean arterial” OR “Blood pressure” OR “Arterial pressure” OR “Systolic pressure” OR “Diastolic pressure” OR “Pulse pressure” OR “Venous pressure” OR “Pressure monitor” OR “Prehypertension” OR “BP”“Tai Ji” OR “Tai-ji” OR “Tai Chi” OR “Tai Chiquan” OR “Qi gong” OR “Baduanjin” OR “Eight trigrams boxing” OR “Yoga” OR “Yogic” OR “Asana” OR “Pranayama” OR “Meditation” OR “Football” OR “Soccer” OR “Swim” OR “Natation” OR “Aquatic” OR “Brisk walking” OR “Walk” OR “Run”“Randomized Controlled Trial” OR “Clinical Trial” OR “Randomized Clinical” OR “Random Allocation” OR “Randomization” OR “RCT”#1 AND #2 AND #3We searched the candidate articles in the PubMed, Web of Science, Embase, and Cochrane Library electronic databases for studies published before November 6, 2020. The following search terms were used in the databases:Based on the PICOS principle of the Cochrane Handbook, the inclusion and exclusion criteria were determined, and the literature was strictly screened. The inclusion criteria of this meta-analysis were as follows: (1) The subjects were defined as patients with a diagnosis of hypertension. (2) The six types of exercise, walking, yoga, aquatic sports, football, Tai Chi, and Qigong, had corresponding controls. (3) Blood pressure values were reported after a prescribed period of exercise intervention. (4) The study reported the blood pressure and standard deviation of patients after the exercise intervention or changes in blood pressure and standard deviation before and after the intervention. (5) The study design was a randomized controlled trial. Two reviewers independently retrieved the papers and imported them to Endnote X9 to manage the search records. Screening of the titles and abstracts of the search records was conducted based on the inclusion criteria. The full texts of the eligible articles were also retrieved for further confirmation, and relevant data were extracted from the selected studies, including the first author name, types of exercise, country, number of participants, ages, sex, intervention, control, frequency, duration of the intervention, and outcome measures.The risk of bias in the included studies was assessed according to the Cochrane Handbook version 5.3.0. The risk of bias assessment was independently completed by two reviewers, and any conflict was resolved by a third reviewer.I2 statistics were used to test the heterogeneity between the studies. An I2 ≥ 50% was considered to indicate high heterogeneity across the included studies, and the random-effects model was adopted for the corresponding effect value combination; otherwise, the fixed-effects model was adopted [P < 0.05 was considered significant.Since our data units were consistent, the combined results were expressed in weighted mean differences (WMDs) and 95% confidence intervals (CIs). There were two forms of mean blood pressure values, postintervention values and change values . When the baseline blood pressure value was known, we tended to calculate the change because the change eliminated individual differences and was more comparable.  adopted . A positA total of 5,452 candidate papers were initially identified. After reviewing the titles and abstracts, 103 studies were considered for further review. Of these studies, 15 were not RCT designs, 19 interventions were unclear, 12 studies were not conducted in a hypertensive population, and 11 focused on other outcome measures. Finally, 46 studies met the inclusion criteria for our meta-analysis. The detailed selection process is shown in A total of 3,058 hypertensive patients from 14 regions were included, and the sample size ranged from 18 to 770. The average age of the confirmed patients ranged from 37 years to 70.24 years. The duration of the interventions ranged from 2 weeks to 16 months. There were 11 studies that used walking as an intervention measure, which had the largest number of included studies, including 762 patients. There were 5 studies using football as an intervention, which had the least number of included studies, including 150 patients (79 in the intervention group and 71 in the control group). 25 studies used no treatments or no physical activity as control measures, while control groups in other studies received wellness education, standard lifestyle modifications, and standard clinical care. The characteristics of all included studies are shown in The quality assessment checklist was used to ensure that the included studies met the quality requirements. The results of the risk of bias assessment are shown in Eleven studies reported associations between walking and blood pressure changes, involving 762 hypertensive patients, with 442 in the experimental group and 320 in the control group. The pooled effect value showed that systolic blood pressure in the experimental group was reduced by 5.94 mmHg  compared with the control group, and the difference was statistically significant. Similarly, the diastolic blood pressure of the walkers was 2.66 mmHg lower than that of the control group  .Ten studies focused on the effect of a yoga intervention in hypertensive patients by randomized controlled trials. The results of the meta-analysis showed that, compared to the control group, hypertension patients who underwent the yoga intervention had significant benefits in lowering their blood pressure, with systolic blood pressure decreasing by 5.09 mmHg  and diastolic blood pressure decreasing by 3.06 mmHg  .A total of nine studies compared the blood pressure of patients in the two groups after an aquatic sports intervention. A random-effects model was used for meta-analysis. The results showed that the improvement in systolic  and diastolic  blood pressure in the aquatic sports group was better than that in the routine nursing group, and the difference was statistically significant .Football is also one of the most common aerobic exercises, and five studies with football training interventions for people with high blood pressure were included, with three from the UK and the remaining two from Denmark and Sweden. Metaresults showed that the systolic blood pressure and diastolic blood pressure were significantly reduced in the patients with the soccer training intervention  .Six studies were included, and no significant effect of Tai Chi on blood pressure reduction was found for either systolic or diastolic blood pressure . However, we also found that there was a high degree of heterogeneity between the studies, which may be due to the large gaps in the sample size of each study, the length of the interventions, and the implementation of the intervention measures .The five included studies included 131 patients receiving a Qigong intervention and 141 controls. The results of the meta-analysis showed that the blood pressure of the hypertensive patients receiving the Qigong intervention did not significantly decrease, which was consistent with the results of the Tai Chi intervention .  . Funnel P=0.124) .In this study, we systematically reviewed the available evidence from the RCTs and conducted a meta-analysis to explore the effects of different types of exercise interventions on blood pressure improvement in patients with hypertension. Our results showed that walking, yoga, aquatic sports, and football had significant effects on blood pressure reduction. However, Tai Chi and Qigong did not significantly reduce blood pressure, and the results should be interpreted with caution because of the high heterogeneity of the meta-analysis.Hypertension affects human health. There are many types of aerobic exercise, but which type is more effective and the underlying mechanisms remain to be explored. Our analysis showed that walking, yoga, aquatic sports, and football might lead to significant improvements in blood pressure in people with hypertension. Cohen et al. mentioned that yoga can promote mind-body integration and reduce stress, thus affecting blood pressure . Some stQigong has been used in the clinical setting for many years, but there is a dispute about whether it is effective. According to our findings, we cannot conclude that Qigong has positive therapeutic significance for patients with hypertension. Cheung et al. documented that the impact of Qigong on blood pressure is approximately 2 mmHg, and it has no clinical significance . FurtherAerobic exercise is physical exercise of low to high intensity which depends primarily on the aerobic energy-generating process  such as Consequently, aerobic exercise was broadly recommended by various hypertension guidelines, such as Canada's 2018 hypertension guidelines , the JapThe proposed mechanisms of aerobic exercise on blood pressure control include neurohumoural, vascular, and structural adaptations . AerobicAerobic exercise can modulate the renin-angiotensin system (RAS), which is a dual system with two opposite arms . The firIt is widely known that both heritable and lifestyle risk factors contribute to hypertension. In 2018, Evangelou reported that a total of 901 loci were associated with blood pressure , and theOur study has several strengths. First, to the best of our knowledge, this is the first systematic review of blood pressure changes in hypertensive patients with different exercise interventions. Second, many types of aerobic exercise and a large number of participants were included, which increased the accuracy of the effect estimate. Finally, the research design that we included was a randomized controlled trial, which had the highest level of evidence compared with other research design types, so the corresponding effect value combination results were more convincing. Some limitations of our study should be noted. First, our results showed a high heterogeneity level across the included studies, which may be due to the intervention periods and patients' ages. At the same time, the methodological differences in the included studies also contributed to the heterogeneity, and some studies did not blind the subjects and/or the outcome assessors, which may reduce the power of the results. In addition, due to the lack of a detailed description of exercise intensities and frequencies in the included studies, these key exercise principles were not taken into account in our analysis. Finally, the protocol in this study was not registered, and the evidence quality was not evaluated with the GRADE approach, which might affect the explanatory power of the results.Our meta-analysis suggested that exercises such as walking, yoga, aquatic sports, football, Tai Chi, and Qigong were feasible and independent lifestyle interventions, and they were effective options for treating hypertension. Despite these encouraging findings, more scientifically designed randomized controlled trials are needed to further compare different forms of exercise for the treatment of hypertension to provide evidence-based recommendations for different types of exercise as antihypertensive treatments.


The useability of DPIs  depends on several factors that are influenced by the patients’ subjectivity and objectivity. The short-form global usability score (S-GUS), a specific tool for the quick ranking and comparison in real life of an inhaler’s usability, was used to investigate six of the most prescribed DPIs  in consecutive asthma patients aged <18 years. A Bayesian indirect comparison (IC) was carried out to merge all pairwise comparisons between the six DPIs. Thirty-three subjects participated: eighteen tested Breezhaler, Spiromax, Nexthaler, and Ellipta simultaneously, while fifteen tested Breezhaler, Spiromax, Diskus, and Turbohaler. The estimates of the S-GUS, by the IC model, allowed us to rank the DPIs by their degree of usability: Ellipta, Diskus, and Spiromax were classified as “good to pretty good” (S-GUS > 15), while Spiromax, Turbohaler, and Breezhaler were classified as “insufficient” (S-GUS < 15). The multidomain assessment is recommended in asthma adolescents in order to approximate the effective usability of different DPIs as best as possible. The S-GUS proves particularly suitable in current clinical practice because of the short time required for its use in adolescents. The adherence to inhalation therapy, together with the personalization of respiratory treatments, have been strongly supported in the last two decades for persistent bronchial asthma needing the long-term use of active drugs, and particularly in subjects prone to insufficient compliance to inhalation, such as the elderly and adolescents ,2,3. Dry powder inhalers (DPIs) became progressively available as a result of technological engineering ,5,6. HowThe factors affecting patient adherence to inhalation treatments via DPIs have been extensively studied. In particular, they have been mostly investigated from the point of view of patients, in terms of their preference, acceptance, and satisfaction ,13,14,15The global usability score (GUS) is an anonymous questionnaire specifically developed for objectively assessing the usability of inhalation devices . The GUSThe study aimed to assess and compare the real-life usability of six of the most used DPIs in asthma adolescents.p < 0.001) [The short-form GUS questionnaire (S-GUS) was preferred for the present study because, while maintaining the same sensitivity of the extensive version, it is much faster to fill  , and it < 0.001) , the meaThe S-GUS questionnaire, reported in 0.5 L/min) [The six DPIs to compare were: Breezhaler, Diskus, Ellipta, Nexthaler, Spiromax, and Turbohaler. These DPIs were selected because they are characterized by different intrinsic resistances (ranging from 0.017–0.039 kPa5 L/min) ,22, and The methodology was previously described . Brieflyp-value lower than 0.05 was considered to indicate evidence of differences in the evaluated variables.The patient characteristics were reported as absolute and relative frequencies; age was the only continuous variable collected in the questionnaire, and it was summarized as the mean and standard deviation (SD). The nonparametric Wilcoxon test was used to check the differences in the age distribution between the subgroups of patients who tested the different sequences of DPIs, while the Fisher’s exact test was used for all the other variables. The analyses were conducted using the statistical software R version 3.6.3 , and a ij,s for Patient s; s are random terms that captures the heterogeneity among patients . The relative device usability, A Bayesian indirect comparison (IC) was conducted to merge all pairwise comparisons between the six DPIs reported in the 33 questionnaires. The model was developed by using the software package, WinBUGS 1.4.3  , and was4) for the device usability and uniform distributions, with supports between 0 and 5 for all the standard deviations. All the simulations were conducted using one chain of 100,000 iterations, and the estimates were calculated after a 50,000-run burn-it. The parameters of interest were summarized by the posterior mean and the 90% credibility intervals (CrI), i.e., the 5th and 95th percentiles of the simulated values. Noninformative vague priors for all the hyperparameters defined in the model were used: normal distribution centered in zero with a large variance (102), according to the classical definition of Higgins and Thompson [The Bayesian approach allows for establishing an efficacy ranking among the analyzed interventions. The surface under the cumulative ranking (SUCRA)  was calcThompson .A total of thirty-three consecutive asthma adolescents were included in the study: eighteen patients (Group 1) tested Breezhaler, Spiromax, Ellipta, and Nexthaler, and fifteen patients (Group 2) tested Breezhaler, Spiromax, Turbohaler, and Diskus. The general characteristics of the sample are reported in p = 0.107). Group 1 and Group 2 were similar for almost all collected characteristics . The proThe GUS values estimated for each device are summarized in According to the thresholds previously proposed , Ellipta2 = 67.4%). Finally, the S-GUS score values proved large indeed, with a total heterogeneity  equal to 24.81. However, the patients’ characteristics seem to explain almost 70% of the variability  and objective  domains. It should be considered as the discriminating parameter for choosing the proper DPI, as it defines the overall “real-word” convenience of each DPI. This was the main reason for adopting the multidimensional GUS questionnaire in the present study on asthma adolescents. Even if all the DPIs tested in the present study did not achieve the GUS top score of 50 points, their usability proved clearly ranked in asthma adolescents, simplifying their choice in real life. Moreover, the patients’ usability elaborated in the present study is consistent with those observed in a cohort of asthmatic adults previously instructed in the use of DPIs . The ranThe present study has some limitations. The study is a monocentric investigation with a small sample size. Each patient did not test all the selected DPIs simultaneously, but only four-by-four. On the other hand, this was decided on purpose because the simultaneous comparison of more than four DPIs would have affected the reliability of the patient responses. Actually, despite the strict control of the nurses’ explanations of each DPI procedure, it was possible that minimal differences occurred while delivering the educational messages. DPI usability is confirmed as a complex issue indeed. DPIs are characterized by different degrees of usability in asthma, and also in adolescents. One single parameter only, based on the patient’s perceptions and beliefs, is unable to exhaustively represent per se the usability of DPIs, and allow for the most effective choice. A multidimensional score should be preferred in asthma adolescents in order to approximate the effective usability of the different DPIs presently available at the best possible level. The current conviction that “one size fits all” is clearly misleading in clinical practice when using DPIs, particularly in asthma adolescents. The opportunity to quickly rank the usability of DPIs should be regarded as a value, and should then be carefully considered before prescribing or switching a DPI in asthma adolescents.


In primary care (PC), 80% of the acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are treated. However, no predictive model has been derived or validated for use in PC to help general practitioners make decisions about these patients.To derive a clinical prediction rule for mortality from any cause 30 days after the last PC visit.Between December 2013 and November 2014, we performed a cohort study with people aged 40 and over who were treated for AECOPD in 148 health centres in Spain. We recorded demographic variables, past medical history, signs, and symptoms of the patients and derived a logistic regression model.In the analysis, 1,696 cases of AECOPD were included and 17 patients (1%) died during follow-up. A clinical prediction rule was derived based on the exacerbations suffered in the last 12 months, age, and heart rate, displaying an area under the receiver operating characteristic curve of 0.792  and good calibration.This rule stratifies patients into three categories of risk and suggests to the physician a different action for each category: managing low-risk patients in PC, referring high-risk patients to hospitals and taking other criteria into account for decision-making in patients with moderate risk. These findings suggest that it is possible to accurately estimate the risk of death due to AECOPD without complex devices. Future studies on external validation and impact assessment are needed before this prediction rule may be used in clinical practice. KEY MESSAGESIn primary care, 80% of COPD exacerbations are attended.Short-term death due to COPD exacerbation can be accurately predicted in primary care without any complex instrument.Further research is needed to perform an extensive validation study in primary care for this and similar predictive models.Chronic obstructive pulmonary disease (COPD) is the fourth most frequent cause of death in the world. It is expected that mortality from COPD will continue to worsen in the coming decades, mainly due to the increase in tobacco consumption in low- and middle-income countries .Many exacerbations occur throughout the life of a person with COPD, which the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines define as ‘an acute worsening of respiratory symptoms that result in additional therapy’ . Until rA subgroup of people with COPD suffer frequent exacerbations: those with two or more exacerbations per year. These people suffer a faster deterioration of lung function, longer time at home, more inferior quality of life, higher probability of hospital admission, and higher risk of death than those with fewer exacerbations, regardless of the degree of deterioration of their lung function . The besExacerbations of COPD are heterogeneous. Lower respiratory inflammation is different depending on whether the aetiology is viral or bacterial infections. Also, patient characteristics affect the severity of the exacerbation. Decision-making in this context is complex and a tool to help physicians would be useful for both doctors and patients .As expressed in the GOLD guidelines, ‘prevention, early detection, and prompt treatment of exacerbations are vital to reduce the burden of COPD.’ Although some predictive models have been published, they were derived and validated in the hospital setting. Almost all these models include variables that cannot be assessed in primary care (PC) . We hypoMethods have been described in detail elsewhere . We desiTo study the prognosis of the AECOPD episode as a whole and not the prognosis of each visit to the general practitioner (GP) that the patient made during the same episode, we considered the visits made in the four weeks after a visit for AECOPD as part of the same episode of AECOPD. For patients who had several values of the same variable during the same episode of AECOPD, we selected the value corresponding to the visit in which the GP established that the patient had a worse general condition. In patients who made several visits, death was determined 30 days after the last visit. In patients who had several exacerbations, each of them was considered as an independent exacerbation.2), systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), peripheral temperature, oedema in the legs, confusion, grade of dyspnoea according to the modified dyspnoea scale of the Medical Research Council (mMRC) scale, Charlson comorbidity index, cardiovascular disease, diabetes mellitus, dementia, cancer , being included in the home care programme, type of health centre , season in which the episode began, and the number of exacerbations registered in the EHR in the last 12 months. The last body mass index (BMI) value was also included if it had been registered within the last year, and the last percentage between the observed and the expected forced expiratory volume in one second was included if it had been registered in the last two years.The outcome was death from any cause within 30 days after the last visit due to AECOPD. Evaluation of the independent variables was performed without knowing the result of the outcome. Independent variables were, at the time of the visit, sex, age, peripheral arterial oxygen saturation  and people with significant mobility impairments  regardless of the cause, provided that the foreseeable duration of this disability exceeds two months.χ2 test in the rest of the cases. The effect of quantitative variables was studied with the Mann–Whitney U test after studying its normality with the Kolmogorov-Smirnov and Shapiro–Wilk tests in both individuals who died and those who did not.The descriptive study of the sample was carried out by a table of frequencies for the qualitative variables and a table of medians and interquartile ranges for the continuous variables. In the univariate analysis, the effect of qualitative variables was studied with Fisher's exact test when the expected frequency was less than five in more than 20% of the cells and with the 2, SBP, DBP, HR, BMI, and peripheral temperature, so that the values of the imputations were biologically plausible. The type of univariate model type used was multinomial logistic regression for categorical variables and linear regression for scale variables. Hundred imputations were made because the proportion of missing data was high. The SPSS syntax used can be found in the supplementary information.We carried out descriptive and pattern analyses of the missing data in which all the independent variables and outcomes were included. Assuming that missing data were missing at random, we performed a multiple imputation procedure in which the outcome and all the independent variables were included, using a fully conditional specification method with ten maximum iterations. Minimum and maximum allowable imputed values were defined for SpOWe excluded independent variables whose predictive effect had not been demonstrated in previous studies and was not suspected based on the clinical knowledge of the principal investigator. Sex was excluded based on results of the univariate analysis. For categorical variables, categories with few elements were collapsed to maximise their statistical significance in univariate analysis. For continuous variables, extreme outliers, defined as values more than three times the interquartile range below the first quartile or above the third quartile, were truncated. Quantitative variables were not categorised, and their linear relationship with the logit model of the probability of the outcome was studied using the Box-Tidwell test. Only the number of previous exacerbations did not demonstrate linearity, so we iterated some simple transformations for this variable and root square transformation demonstrated linearity. The presence of collinearity between these variables was also studied through the analysis of principal components. Finally, we studied all the possible interactions of age with the remaining predictors by adding their cross-products as well as the interactions between the independent variables with statistically significant relationships.p-value less than 0.157. A variable was considered a confounding factor when by eliminating it from the model, the regression coefficient of another variable changed by more than 10 percent. The internal validity of the model was studied through a bootstrapping resampling simulating 1,000 samples for each of the 100 imputations. We applied a uniform shrinkage factor for the regression coefficients calculated with bootstrapping and re-estimated the intercept based on the adjusted coefficients.We derived a logistic regression model for all-cause mortality at 30 days following a stepwise regression method using the logarithm of the likelihood ratio as a selection criterion. The rule to remain in the model was a The model’s discrimination was studied with the area under the receiver operating characteristic curve (AUROC), and the calibration was assessed with the calibration slope and the intercept. These performance measures were pooled parameters calculated from the 100 imputations.To support the clinician making decisions, the result was returned as a mortality risk category , and a different action was proposed for each category. The thresholds were based on predicted probability quintiles and a decision curve analysis was performed.ad hoc form implemented in the EHR and the Spanish National Death Index. Statistical analyses were performed with IBM SPSS Statistics 24 and RStudio 1.4.1106 for Windows.All the data were collected through an The study protocol was approved by the Burgos Research Ethics Committee (reference CEIC 1185), the Salamanca Research Ethics Committee, the Soria Research Ethics Committee (reference CEIC 1227), the East Valladolid Research Ethics Committee (reference PI-13-115), the West Valladolid Research Ethics Committee (reference PI-13-115) and the Zamora Research Ethics Committee .There were 2,238 exacerbations evaluated in 1,536 people. Of those, 307 (13.7%) exacerbations were excluded because a diagnosis of COPD was not included in the associated EHR, 192 (8.6%) were wholly diagnosed and treated in a hospital, and 43 (1.9%) had no available EHR. Finally, 1,696 exacerbations in 1,054 people (1.6 exacerbations per person) from 148 HC were included in the analysis .The mean age of participants was 76 years and 84% was male, 17 people (1%) died within 30 days after the last PC visit for AECOPD. Of these participants, 15 (88.2%) died due to AECOPD and one died due to end-stage renal disease; in one case, the cause of death could not be recovered. Complete characteristics of the sample and the relationship of the predictors with the outcome in the univariate analysis are presented in In the pattern analysis, we observed that the variables with the most missing data were those related to the physical examination and the dyspnoea grade. In the descriptive analysis, we observed that the distribution of the missing data in all the variables that had them was related to some of the other variables.2) values below 75% and four BMI values above 49.74 kg/m2 were truncated. A lack of linearity was detected for the variable ‘exacerbations in the last 12 months,’ which was corrected by transforming it into its square root. No significant interactions were found between the predictors. A shrinkage factor of 0.921 was applied.Diastolic blood pressure, peripheral temperature, oedema in the legs, diabetes mellitus, cancer, dementia, type of HC, and season in which the episode began were excluded because they did not demonstrate predictive ability in previous studies and were not suspected according to the clinical criteria of the authors. The categories of the variable ‘grade of dyspnoea according to the mMRC scale’ were combined to form a dichotomous variable called ‘dyspnoea grade 4 according to the mMRC scale.’ Likewise, the Charlson index categories were combined to form a dichotomous variable called ‘Charlson index greater than 1.’ All extreme outliers were considered biologically plausible,17 peripheral arterial oxygen saturation , which do not need any complex instrument to be measured. The CPR stratifies patients into three categories of risk based on predicted probability quintiles and suggests to the doctor a different action for each category. Patients with low risk might be followed in primary care. Patients with medium risk might be followed closely if the doctor decides to treat them in primary care. Patients with high risk might be followed very closely or referred to the hospital.2) had also been shown to interact in a very complex manner with different systems; in cases of moderate acute hypercapnia, an increase in heart rate is often observed. Tachycardia may therefore be an early sign of an acute increase in pCO2 prior to the onset of headache, agitation, or a decreased level of consciousness [Age clearly showed a predictive effect in this study; the higher the age of an adult suffering from an acute illness was, the greater their probability of dying in the short term. This finding is also compatible with that of previous studies on the subject . The preiousness .p-value less than 0.157 as the stop rule to exclude a predictor from the model instead of using a value less than 0.05. Despite all the above, the only way to determine the true degree of overfitting in the model presented in this work will be through an external validation study [The study’s main limitation is that our sample size was smaller than required . This sion study .This study’s other significant limitation is the large proportion of missing data, which may have occurred because the study did not intend to change the way the doctors collected information but took advantage of data collected in the EHR from usual clinical practice. To compensate for power loss that this causes, a multiple imputation procedure was applied in patients with many imputations .Only 16% of the people studied were women, and only two deaths were observed among them, which could reduce the predictive performance of the rule in women. This small proportion of women is because they represent only between 22 and 29% of people with COPD in Spain .Concerns may arise using COPD diagnoses from EHRs. Previous validation studies suggest that such diagnoses have minor sensitivity and high specificity so they should not be used in prevalence studies but may be used to study risk factors .As this CPR has not been externally validated, this could only be used in similar patients to its derived ones .None of the similar studies published to date derived or validated its rule in PC, where 80% of AECOPD cases are treated . The rulDiscrimination for predicting death from any cause within 30 days after the last visit due to AECOPD in PC in our study was 0,74  for the simplified B-AE-D index, a validated, independent of lung function long-term mortality index in COPD [This is the first predictive model derived from PC for the risk of short-term death due to AECOPD. Although it is a rare event, it can be accurately predicted from knowing the exacerbations suffered in the last 12 months, age, and heart rate. In addition, the rule suggests a different action depending on the calculated risk.There is enough evidence to design a large validation study in primary care of all existing predictive models. Subsequently, the impact that the best predictive model would have on the results of patient-oriented care compared to usual practices should be studied .Click here for additional data file.Click here for additional data file.Click here for additional data file.