Objective: The objective of this study was to determine the prevalence of end-organ damage in hypertensive patients attending an outpatient consultation.Materials and methods: Patients were selected from an outpatient consultation at a tertiary hospital care center. All patients who consulted between July 2022 and March 2023 were included. Data on demographic characteristics, blood pressure records, hypertension etiology, medication use, and the presence of target organ damage were collected.Results: A total of 73 patients were included in the study, with 34 patients being male (46.6%) and 39 patients being female (53.4%). The mean age of the patients was 49.8 years. Among the cases of hypertension, 14 (19.2%) were classified as secondary arterial hypertension (AH). The most common cause of secondary AH was obstructive sleep apnea (OSA) (42.9%). Approximately 23.2% of patients had documented end-organ damage potentially related to hypertension, with kidney disease being the most frequent . The most commonly prescribed pharmacological classes were angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists .Conclusion: Despite numerous studies and trials on arterial hypertension, it remains a significant contributor to morbidity and mortality, necessitating the continued awareness of its long-term implications. Blood pressure is the most commonly measured hemodynamic parameter due to its easy accessibility, but it only represents a small fraction of the overall state of the systemic vasculature . ArteriaSince hypertension is often asymptomatic, many patients, especially younger individuals, underestimate the importance of this condition in society. Therefore, our main objective is to determine the prevalence of end-organ damage in hypertensive patients attending outpatient consultations in order to raise awareness of this public health issue.We conducted a retrospective study at a single-center tertiary hospital, where we selected patients from a specialized consultation for arterial hypertension conducted by an internal medicine physician. The hospital's criteria for referring patients to hypertension consultations include the presence of hypertension at a young age, suspected secondary causes of hypertension, or difficult-to-control hypertension. The evaluation of the secondary forms of hypertension was carried out based on clinical suspicion, particularly in young patients with evidence of end-organ damage, as well as in cases of refractory or resistant hypertension and progressive deterioration of blood pressure profile and/or renal function despite escalated antihypertensive treatment, or when indicated by objective examination.All patients who sought consultation between July 2022 and March 2023 were included in the study. Patients with incomplete registration data or those who had passed away at the time of data collection were excluded. We collected information on demographic data, blood pressure records, the underlying cause of hypertension, medication usage, and the presence of target organ damage. Verbal informed consent was obtained through telephone communication.Our objective was to characterize the population of patients with arterial hypertension in a specialized hospital consultation, including the assessment of the prevalence of secondary forms of the disease, the incidence of end-organ damage, and the most commonly prescribed pharmacological classes.For a nine-month follow-up period, 73 patients were evaluated in a total of 103 consultations. No patients were excluded. Of the total of 73 patients, 34 were males (46.6%), and 39 (53.4%) were females. The mean age was 49.8 years with a median age of 45 years.Figure We found 31 cases (42.5%) of essential and 14 (19.2%) of secondary AH. The remainder still had no established etiology by the time of data collection. In the group of secondary hypertension, six patients had obstructive sleep apnea (OSA) (42.9%), four had renal artery stenosis (28.6%), two had aldosterone-producing adrenal adenomas (14.3%), one had chronic kidney disease (7.1%), and one had Cushing's syndrome (7.1%).Figure A total of 23.2% (n = 17) had end-organ damage(s) potentially related to AH. Of these events, there was a higher proportion of chronic kidney disease , followed by cerebrovascular events, namely, stroke , cardiac events , and peripheral arterial disease .Table The most used pharmacological class, in monotherapy or in combination, was angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists . In descending order, the remaining classes were distributed as follows: calcium channel antagonists , thiazide diuretics , beta-blocker , mineralocorticoid receptor antagonist , and central adrenergic agonist .AH remains an important cause of morbidity and mortality, highlighting the need for research to discover new pharmacological classes . The risOur sample exhibited a bimodal age distribution, reflecting two extremes of referral motives: young patients requiring the exclusion of potentially curable secondary causes of AH and older patients needing a wider range of therapies for blood pressure control. The strict control of blood pressure, particularly in younger age groups, is of paramount importance as it directly affects the risk of end-organ damage and the patient's quality of life. In our sample, a high percentage of patients showed end-organ damage, highlighting the significance of initiating tensional control at a young age to prevent AH complications.Approximately 90% or more of AH cases have an unknown cause, categorized as idiopathic or primary AH . The remModels exploring the pathophysiology of AH are complex, involving the interaction of extrinsic and intrinsic factors, making it difficult to determine the relative importance of each factor ,6-9. ImmThe central role of angiotensin II and aldosterone in blood pressure regulation and as mediators of target organ damage makes the renin-angiotensin-aldosterone system a particularly promising therapeutic target ,6,7. In Certain secondary forms of arterial hypertension, such as primary aldosteronism, are associated with a significantly higher overall risk of vascular complications ,8. AccelOur study has several limitations that must be considered. Our study is retrospective, which means that it relies on past data. This may introduce biases and limitations in data collection and analysis, as it is challenging to control for confounding variables that may influence the results. Our study included a relatively small sample size of 73 patients, which may limit the generalizability of clinical findings. Additionally, the study was performed at a single tertiary hospital care center, which may not fully represent the broader population of hypertensive patients. As we previously mentioned, patients were specifically selected from a specialized consultation for arterial hypertension, which may introduce selection bias and influence the prevalence of end-organ damage. The study data collection period spanned from July 2022 to March 2023, which might not fully capture seasonal variations or long-term trends in hypertension and end-organ damage. We only presented data on the most prescribed drugs, potentially missing important information about medication adherence and patient response to treatment. The study did not account for potential confounding factors , which might influence the observed associations between hypertension and end-organ damage.In conclusion, arterial hypertension (AH) remains a significant health issue, emphasizing the need for research to discover new pharmacological classes. Uncontrolled blood pressure increases the risk of target organ damage over time, underscoring the gravity of this condition. Strict blood pressure control, especially in younger patients, is crucial to reduce end-organ damage, which can have profound implications for patients' quality of life and long-term health outcomes. Secondary forms of AH, although less prevalent, require specialized consultations for accurate diagnosis and treatment. Understanding the complex pathophysiology of AH, including immune mechanisms and cytokine release, highlights the importance of reducing inflammation to prevent end-organ failure. The renin-angiotensin-aldosterone system is a promising therapeutic target, and agents blocking this system are effective in blood pressure control and organ protection.Moreover, primary aldosteronism, a secondary form of AH, warrants a more aggressive management approach due to its association with higher vascular complications, further underlining the significance of tailored interventions. Surgical interventions may be considered earlier in this subgroup to prevent or mitigate potential end-organ failures. Overall, strict blood pressure control and targeted therapies are essential in managing AH and improving patient outcomes. Future research may benefit from expanding the dataset to include additional demographics and exploring links between secondary diseases such as obstructive sleep apnea (OSA) and hypertension while ensuring that the conclusion aligns with the stated study objectives and considers the prevalence of end-organ outcomes as outlined in the objectives.


This study was designed to investigate the controversy on the potential role of sKlotho as an early biomarker in Chronic Kidney Disease–Mineral Bone Disorder (CKD-MBD), to assess whether sKlotho is a reliable marker of kidney α-Klotho, to deepen the effects of sKlotho on vascular smooth muscle cells (VSMCs) osteogenic differentiation and to evaluate the role of autophagy in this process. Experimental studies were conducted in CKD mice fed a normal phosphorus (CKD+NP) or high phosphorus (CKD+HP) diet for 14 weeks. The patients’ study was performed in CKD stages 2–5 and in vitro studies which used VSMCs exposed to non-calcifying medium or calcifying medium with or without sKlotho. The CKD experimental model showed that the CKD+HP group reached the highest serum PTH, P and FGF23 levels, but the lowest serum and urinary sKlotho levels. In addition, a positive correlation between serum sKlotho and kidney α-Klotho was found. CKD mice showed aortic osteogenic differentiation, together with increased autophagy. The human CKD study showed that the decline in serum sKlotho is previous to the rise in FGF23. In addition, both serum sKlotho and FGF23 levels correlated with kidney function. Finally, in VSMCs, the addition of sKlotho prevented osteogenic differentiation and induced autophagy. It can be concluded that serum sKlotho was the earliest CKD-MBD biomarker, a reliable indicator of kidney α-Klotho and that might protect against osteogenic differentiation by increasing autophagy. Nevertheless, further studies are needed to investigate the mechanisms of this possible protective effect. Chronic Kidney Disease (CKD) is a state of decreased kidney function characterized by vascular and bone disturbances, known as Chronic Kidney Disease–Mineral Bone Disorder (CKD-MBD) . The proThe disturbances in calcitriol, P and calcium (Ca) homeostasis are late events in the course of CKD-MBD; on the contrary, the increase in FGF23 and the changes in sKlotho occur in earlier stages of CKD ,5. FGF23Vascular impairment in CKD is often diagnosed when the damage is already stablished and no early biomarkers are known ,16. The This study was designed to (a) confirm the potential role of sKlotho as an early biomarker compared with other CKD-MBD biomarkers, (b) to assess whether sKlotho is a reliable indicator of kidney α-Klotho content, (c) to deepen the effects of sKlotho on vascular smooth muscle cells (VSMCs) osteogenic differentiation and (d) to evaluate if autophagy could be involved in the actions of sKlotho.In the study, three different models were used: (A) mice with mild CKD, (B) patients with CKD stages 2–5 and, (C) vascular smooth muscle cells (VSMCs).n = 13) underwent 75% kidney mass reduction  using two-step surgical intervention. The first intervention consisted of the decapsulation of the kidney and afterwards a bipolar resection of the left kidney by cauterization. One week later, the right kidney was totally removed. The anesthetic method used was isoflurane by inhalation. One week after the second intervention, they were divided into two experimental groups: One group received a normal phosphorus (NP) diet  and the other group received a high phosphorus (HP) diet . A third group of Sham-operated mice fed an NP diet (n = 8) was used as reference group. All mice received the described food for 14 weeks, thereafter they were placed in metabolic cages for 24-h urine collection prior to sacrifice. At sacrifice, blood and urine samples, aortas and kidneys were collected and stored at −80 °C until analysis.To induce CKD, two and a half-month-old male FVB/N mice .Blood chemistries’ analysis utilized Quanti-Chrom™ Specific Assay Kits (Bioassay Systems) for serum creatinine, BUN, Ca and P. Specific ELISA kits quantified the serum intact parathyroid hormone , intact FGF23 , calcitriol 2D3; Elabscience Biotechnology Inc., Houston, TX, USA) and sKlotho .In urine samples, Quanti-Chrom™ Specific Assay Kits  were used to measure Ca and P, and ELISA assays were performed to quantify albumin  and sKlotho levels .Kidney interstitial fibrosis was determined using Masson’s Trichrome staining (standard protocol) in deparaffined 2.5 µm sections of the kidney. It was quantified by using an optical microscope  coupled to a digital video camera  and to an image analysis system with specific software (ImageJ 1.53k). Measurements were performed by a technician blinded to experimental groups and interventions. Results were expressed as the percentage of blue area of the total kidney area excluding peri-glomerular and peri-vascular blue staining.The expression analysis by immunohistochemistry of kidney α-Klotho and non-phospho (active) β-catenin were performed using the EnVision FLEX Mini Kit  and Dako Autostainer systems. Paraffin-embedded kidneys (2.5 µm) were deparaffinized, rehydrated and underwent epitope retrieval (HIER) by heat induction at 95 °C for 20 min and pH 9  in the Pre-Treatment Module, PT-LINK . Kidney sections were incubated with Klotho and non-phospho (active) β-catenin antibody  diluted Two methods were used to assess soft tissue Ca deposition: Von Kossa  and AlizAortic paraffin sections of 2.5 µm were incubated overnight with anti-LC3BII antibody  at 4 °C A total of 43 CKD patients (25 men and 18 women) and 38 sex- and age-matched controls (18 men and 20 women) were studied. The average age was 66.65 ± 8.58 in CKD patients and 66.50 ± 4.63 in controls. The CKD patients were classified according to their estimated glomerular filtration rate (eGFR) into four groups: CKD-2/3a, CKD-3b, CKD-4 and CKD-5 [Age, sex, body mass index (BMI), blood and urinary parameters were collected. Creatinine was determined by isotope dilution mass spectrometry . Total proteins, Ca and P were determined by photometry , PTH using an electrochemiluminescence immunoassay  and FGF23 and calcitriol were analyzed with a chemiluminescence immunoassay . Finally, the concentration of sKlotho, both in serum and urine, was evaluated by a specific ELISA assay .Two different in vitro approaches were used (i) to analyze the possible effect of sKlotho on VSMCs muscular and osteogenic phenotype changes towards calcification after 24 and 72 h of incubation in calcifying (CM) and non-calcifying medium (non-CM) and (ii) to study if autophagy plays a role in the effect of sKlotho on the above mentioned VSMCs’ phenotypic changes. Primary VSMC cultures were obtained from aortas of normal non-manipulated 2-month-old Wistar rats using explants as previously described ; cells b2 in 6-well plates  and grown in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin  to sub-confluence. Cells were exposed to Non-CM , CM  [VSMCs were seeded at 100,000 cell/cm 3 mM P) ,28,29 or 3 mM P) ). This r 3 mM P) ,31 and e 3 mM P) . The expTo characterize if sKlotho induces autophagy, primary VSMCs and A7r5 (ATCC) cells were seeded in 96-well imaging plates  for immunofluorescence and in 6-well plates to obtain protein for Western blot.The primary VSMCs and A7r5 cells were exposed during 24 h to Non-CM, CM and CM+sKlotho. To induce autophagy, after 20 h (4 h before the end of the experiment), cells were exposed to nutrient free media (Nf). Immunofluorescence (LC3B-II puncta) and Western blot   in Tris-buffered saline with Tween 20 buffer (TBST) for 30 min at room temperature and incubated with anti-LC3B  diluted Total RNA from kidney, aorta and primary VSMCs was extracted using TRI reagent (Sigma-Aldrich) and 2µg of total RNA was retro-transcribed using the High Capacity cDNA Reverse Transcription Kit . Quantitative real time PCR (qPCR) was performed using pre-developed assays  in the STotal proteins from kidney and primary VSMCs were extracted using a standard RIPA buffer with protease inhibitors and 0.2 mM sodium orthovanadate .For kidney samples, a total of 40 μg of protein was electrophoresed on 8% SDS-PAGE minigels and transferred to a Hybond™ P membrane . Primary antibodies are specified in For primary VSMC, a total of 15 μg of protein sample was electrophoresed on 15% SDS polyacrylamide gels and electro-transferred onto polyvinylidene difluoride (PVDF) membranes . Primary antibodies are specified in p value lower than 0.05 was considered statistically significant. All calculations used the statistical analysis package R (3.6.1 version).Results are expressed as median (interquartile range) or mean ± standard deviation as indicated. Statistical comparisons between groups were performed using the Kruskal–Wallis test  or ANOVA  and Dunn or Tukey tests  as post hoc analysis. A p = 0.0005; BUN: r = −0.573, p = 0.007; Ca: r = −0.453, p = 0.039; PTH: r = −0.549, p = 0.012; FGF23: r = −0.471, p = 0.031 and phosphaturia: r = −0.468, p = 0.037).After 14 weeks, both groups of nephrectomized mice (CKD+NP and CKD+HP) showed significantly higher serum creatinine and BUN levels compared to the reference group (Sham-operated mice+NP), without differences between the CKD groups. In addition, the CKD+HP mice showed lower body weight, higher serum levels of P, PTH and FGF23 and lower levels of serum sKlotho . A negatIn the urine, the CKD+HP group showed lower levels of Ca and sKlotho and higher levels of P and urinary albumin compared to the CKD+NP group . There wp = 0.0007; p = 0.00007). No correlation was found when the reference group was excluded . The kidney collagen 1 gene expression followed a similar pattern achieving the highest mRNA levels in the CKD+HP group . In the CKD groups, urinary sKlotho decreased (p = 0.40).Kidney mRNA α-Klotho expression was significantly lower in the CKD+HP group compared to the reference and the CKD+NP groups A. Klothoecreased , but it p = 0.327), but Dkk1 levels were significantly higher in the CKD+HP group compared to the CKD+NP group .The kidney Wnt/β-catenin pathway signaling showed a significant activation in the CKD+HP group A, which p = 0.027, A significant lower vascular α-actin A and Sosp = 0.004).Aortic Ca deposition was not observed in any group. The kidney showed Ca deposits in the two CKD groups having a significant negative correlation with kidney α-Klotho protein .No changes were observed in serum Ca and serum P showed a significant increase in CKD-5 . Serum PThe urinary sKlotho significantly increased in CKD-2/3a stage  and it rp = 0.002). The addition of sKlotho (50 ng/mL) did not modify the α-actin levels in the CM group .After 24 h of exposure of the primary VSMCs to CM and non-CM, no changes in Ca content, Osterix and Sost gene expression were observed and α-actin gene expression was significantly lower in the VSMCs cultured in CM . The cellular content of LC3B puncta was significantly greater compared to VSMCs exposed to CM without sKlotho A,B.The measurement of the autophagic flux by the incubation with Bafilomycin A1 during the last 4 h of CM+sKlotho exposure, led to an increase in LC3B-II cellular levels comparable to that observed in the positive control medium (Nf) C–E. The This study was designed to (a) confirm the potential role of sKlotho as an early biomarker of CKD-MBD, (b) to assess whether sKlotho is a reliable indicator of kidney α-Klotho content, (c) to deepen the effects of sKlotho on VSMCs osteogenic differentiation and (d) to evaluate if autophagy could be involved in the actions of sKlotho.The in vivo experimental study showed that serum sKlotho was a reliable marker of kidney α-Klotho. In addition, it showed a significant decrease in sKlotho before the FGF23 increased, which was more pronounced in the CKD mice exposed to a high P diet (CKD+HP). In the aortas of the mice, serum sKlotho correlated with the loss of α-actin as a marker of the VSMCs contractile phenotype. The study in CKD patients confirmed that serum sKlotho decreased early during the course of CKD, and in the same way as in the mice study, this happened even before FGF23 levels started to rise. Finally, the in vitro study showed that sKlotho mitigated the VSMCs osteogenic differentiation and increased autophagy.Klotho is synthesized mainly in the kidney, although it has also been detected in other tissues . CleavagThe mice model showed that after 14 weeks of a mild-moderate CKD (CKD+NP group), creatinine and PTH experienced a two-fold increase, which coincided with a significant reduction in serum but not urinary sKlotho. Importantly, the decrease in sKlotho occurred before the changes in other CKD-MBD biochemical parameters, such as Ca, P, calcitriol and even FGF23. Coincident with previous studies ,39,40, tUrinary sKlotho levels decreased in both CKD mice groups following the same pattern of serum sKlotho, though the magnitude of the changes was lower than those observed in serum sKlotho. Importantly, urinary sKlotho decreased, but it did not correlate with kidney α-Klotho content, in contrast with the highly significant correlation observed between serum sKlotho and kidney α-Klotho D. These The reduction in serum and urinary sKlotho observed in the CKD groups coincided with high percentages of kidney fibrosis compared with controls, which goes in the same direction as other studies which showed that Klotho protected against renal fibrosis in mice ,45. In sAs kidneys are the main source of sKlotho, but at the same time the reduction in renal function can impair sKlotho removal in CKD, the interpretation of the values of urinary sKlotho is controversial ,14. sKloThe study of patients with different CKD stages confirmed that serum sKlotho was the earliest CKD-MBD biomarker that showed changes and it was also observed in the experimental study, serum sKlotho decreased before FGF23 started to increase A. The clIn CKD patients, urinary sKlotho followed an opposite pattern than serum sKlotho. It started to change in early stages of CKD, but it increased instead of decreased. A possible explanation for the discrepancy with the results in mice could be due to the different chronology, type and degree of renal insufficiency of mice and patients. The surgical experimental model of kidney damage may have proportionally affected more, or in a different way, the tubular structures compared with the spontaneous pathophysiological CKD alterations that occur in patients, in whom different glomerulo-tubular changes could be expected. The surgical-dependent morphological and structural kidney changes induced by the surgery in the mice model, may partly explain the difficulties in the tubular processing of sKlotho, and therefore impairing its removal. On the other hand, the increased excretion of sKlotho observed in the CKD patients may have favored the decrease in sKlotho observed in serum. Nevertheless, there are other studies that found no correlation between serum and urine sKlotho .The results of the present study favor the practical value of serum compared to urinary sKlotho to predict α-Klotho kidney content, despite other studies having reported opposite results ,10,47. AIn addition, so far, there is no consensus about what it is the best technique to assess sKlotho, some authors claimed that sKlotho immunoprecipitation might be a better option than performing an ELISA assay, but the labor-intensive nature of immunoprecipitation requires further research, and it might not be extended to all laboratories . In thisIt is important to stress that the strain of mice FVB/N used in our study is considered an appropriate mice model to study kidney damage ,51, but Furthermore, despite the CKD mice not developing aortic calcification, important molecular changes related with the VSMCs changes that occur before VC is established were observed. In fact, the CKD groups showed lower aortic gene expression of Sost and α-actin, indicating a progressive loss of the VSMC contractile phenotype. In addition, α-actin positively correlated with serum sKlotho C, supporsKlotho has been involved in the regulation of the Wnt/β-catenin signaling , and it Autophagy has been proposed as a defensive mechanism against VC in CKD ,22 and sUnfortunately, as autophagy is an extremely fast process , it was The in vivo and in vitro effects of sKlotho found in our study are in keeping with other effects attributed to Klotho such as pro-autophagic actions in heart and kidneys , renoproThe present study has limitations. Despite the aggressive surgery carried out for obtaining renal damage in the rodent model, the level of CKD obtained might be assimilated to a CKD 3a-3b stages in humans. More specifically, CKD+NP mice could be said to mimic the human CKD 3a stage and the addition of a HP diet aims to increase the damage to mimic a CKD 3b stage. Thus, CKD+HP mice displayed a higher kidney damage and vascular impairment, discussed in previous paragraphs. However, it is necessary to take into account the inaccuracy to extrapolate the results obtained in an experimental model in mice to what is happening in the human CKD. Thus, the CKD mice and patients’ studies seem to be only partially extrapolated, as the spontaneous loss of renal function in CKD patients and the surgical reduction performed in the mice could have had different impacts on the mechanisms of sKlotho tubular removal ,43. MoreIn summary, the present clinical–experimental study showed that serum sKlotho declined early, before FGF23 increased, and it was a precise and reliable marker of the kidney α-Klotho content. In addition, the in vitro experiments showed that sKlotho recovered the vascular phenotype, reduced the osteogenic differentiation of the VSMCs and increased the autophagic signaling.


O-methyltransferase (COMT) genotype may play an integral role in blood pressure status and thereby cardiovascular health. This is an area especially important for women who are post-menopause; however, the current literature investigating these associations is limited. This study was a cross-sectional secondary analysis of baseline data (N 237) from the Minnesota Green Tea Trial (MGTT). The current study explored the relationships between plasma adiponectin, leptin, and COMT genotype on blood pressure measures. Plasma adiponectin and leptin were obtained after an overnight fast of at least 10 h and were measured by the radioimmunoassay method. The relationships were analysed using multiple linear regression after adjusting for potential confounders. Effect modifications by age, body mass index (BMI) category, blood pressure category, antihypertensive medication use, and COMT genotype were also investigated. The majority of participants were non-Hispanic (97⋅9 %) and Caucasian (94⋅9 %). Mean (sd) age and BMI were 60⋅7 (5⋅0) years and 28⋅2 (2⋅9) kg/m2, respectively. After adjustment for confounding variables, neither plasma adiponectin, plasma leptin nor COMT genotype was associated with systolic or diastolic blood pressure measures. The results of stratified analyses also did not reveal any significant interactions or associations. Based on the findings of this study, which utilised more rigorous statistical methods than previous research, neither adiponectin, leptin nor COMT genotype play a role in blood pressure measures in women who are post-menopause.Research suggests that adiponectin, leptin, and genetic polymorphisms such as catechol- The aetiology of CVD is multifactorial and has long been associated with comorbid conditions such as diabetes, obesity, and hypertension.–6 Novel research, however, suggests that hormones, such as adiponectin and leptin, and genetic polymorphisms such as catechol-O-methyltransferase (COMT) genotype may also play an integral role in the development of the disease as well.–14Changes that occur as a result of menopause such as withdrawal of oestrogen, increased waist circumference, and other biochemical changes may put women at increased risk of cardiovascular disease (CVD).,16 An inverse relationship between hypoadiponectinemia and hypertension has been previously reported in both men and women who were hypertensive. This relationship may be attributed to adiponectin's effects on the vascular system or implications related to fat or glucose metabolism. While these findings are interesting, gender differences in plasma adiponectin levels also exist that should also be accounted for.,16–18 For example, the majority of research that has specifically explored the relationship between adiponectin and blood pressure in women who are post-menopause does not support the reported findings of an inverse relationship.,19–23Adiponectin is an adipokine or fat-derived hormone that contributes to many important actions in the body and has also been found to play a role in blood pressure through its role in endothelial function, nitric oxide production, and its support for the action of anti-inflammatory macrophages.,24–28 Many studies have demonstrated a positive correlation between leptin and blood pressure.,29–32 Also, similar to adiponectin, gender differences exist with women's leptin levels generally being higher than that of men's leptin levels. Available research exploring the association between leptin and blood pressure in women who are post-menopause suggests that there is a relationship in those ranging from 50 to 68 years old.,22,34 Interestingly, however, leptin has been primarily associated with systolic blood pressure (SBP) in this population.,20,22,34Similar to adiponectin, leptin is an adipokine that may also impact blood pressure status due to its effect on sympathetic nervous system activation at the renal level.O-methyltransferase or COMT is an enzyme that is involved in the metabolism of catecholamines, oestrogens, and catechins.,8,35COMT genotype is polymorphic; an individual's COMT genotype may impact COMT enzymatic activity.,9,35,36 Evidence exists to support a potential relationship between COMTA/A (low activity) genotype and higher SBP, however, to date, no study has explored this association specifically in women who are post-menopause.Catechol-COMT genotype on blood pressure in women who are post-menopause is limited. Understanding more about these novel factors can perhaps help to identify those potentially at risk at an earlier time which could also help with earlier monitoring and potentially contribute to the prevention of CVD as well. This cross-sectional study, therefore, contributes to and expands upon the existing literature with a primary aim to determine the associations between plasma adiponectin and leptin levels and blood pressure among women who were post-menopause. We hypothesised that there would be an inverse relationship between adiponectin and blood pressure while there would be a positive association between leptin and blood pressure in women who were post-menopause. In addition, a secondary and exploratory aim of this study was to determine the associations between COMT genotype and blood pressure among the same sample. We hypothesised that there would be a positive association between COMTA/A genotype and blood pressure while there would be no association between G/G and G/ACOMT genotypes and blood pressure in women who are post-menopause.Current evidence exploring the associations between adiponectin, leptin, and  The MGTT was a randomised double-blind placebo-controlled clinical trial in which the participants were randomly assigned to a green tea extract supplement or placebo group. The primary aim of the parent study was to investigate the effect of the consumption of green tea extract on biomarkers of breast cancer risk in a group of women who were post-menopause, between 50 and 70 years of age, and who were at high risk for breast cancer due to having heterogeneously (51–75 % glandular) or extremely (>75 % glandular) dense breasts.This study was a cross-sectional study, which was completed as a secondary analysis of existing data from the parent study, the Minnesota Green Tea Trial (MGTT).N 237) in the sample. The MGTT was an interventional study where a green tea extract supplement was consumed by the participants; therefore, baseline data was used to exclude any potential effect that the intervention may have had on the variables of interest for this study.,38 Potential participants in the MGTT were identified by study staff reviewing routine screening mammogram reports. Participants were then contacted to identify interest and further included in the MGTT based on the inclusion and exclusion criteria detailed in the following. Participants were included in the MGTT if they were generally healthy women, were post-menopause between 50 and 70 years old with heterogeneously (51–75 % glandular) or extremely (>75 % glandular) dense breasts and required to reside in or near Minnesota during the study. Participants also gave informed consent as part of the inclusion criteria of the MGTT. Participants were excluded if they consumed more than one cup of green tea per week, had Hepatitis B or C, elevated liver enzymes , a history  or use of menopausal hormone therapy during screening, a previous diagnosis of breast or ovarian cancers, a history of any other cancers within past 5 years, proliferative breast disease, breast implants, BMI <18⋅5 or >40 kg/m2, were taking methotrexate or etanercept, were involved in a weight gain or loss programme, had a weight change of greater than 10 pounds during the year prior to recruitment, had an intake of more than seven alcoholic drinks per week, or smoked cigarettes or consumed other tobacco products. The present study utilised the same inclusion and exclusion criteria as the MGTT with two small additions based on the specific outcomes of interest for this study. While there were no additional exclusions added for the execution of this study, participants with available plasma adiponectin and leptin data at baseline of the MGTT was an additional inclusion criterion for this study.The dataset for this study was a subgroup of the population recruited for the MGTT. Only the women who had plasma adiponectin and leptin values available for the baseline visit were included  as defined by the American Heart Association (AHA) and the American College of Cardiology's (ACC) 2017 guidelines were used to assess the data. As the parent study was completed prior to 2017 classifications of the AHA and ACC (normotensive: <140/90 v. hypertensive: >140/90), further stratified analysis was conducted to explore the differences that may exist in the findings based on the different blood pressure guidelines.The outcome of interest for this study was blood pressure measures including both SBP and diastolic blood pressure (DBP). As part of the MGTT, blood pressure was measured using an automated digital vital sign monitor . Whole blood samples were separated into plasma and serum and stored at –80°C until the time of analysis. Adiponectin and leptin concentrations were quantified in plasma utilising the radioimmunoassay method using EMD Millipore kits (inter-assay coefficient of variation (CV): leptin = 7⋅1 %, adiponectin = 8⋅9 %; intra-assay CV: leptin = 6⋅6 %, adiponectin = 7⋅4 %).Plasma adiponectin and leptin levels were measured in blood samples from the MGTT that were obtained after an overnight fast of at least 10 h.COMT genotype was determined from the MGTT using whole blood samples obtained from non-fasting blood draws and analysed by the University of Minnesota Genomics Center.,38 DNA was extracted using the Qiagen DNAeasy Blood and Tissue Kit method .,38 To determine the COMT polymorphism, a TaqMan assay was developed using a TaqMan PCR Core Reagents Kit (Applied Biosystems).,38 Additionally, as a quality control for each PCR run, Coriell cells with a known COMT genotype were used. The data are presented and analysed by the activity of the relevant COMT genotypes: low (A/A), intermediate (G/A), and high (G/G) activity COMT genotypes.2, overweight: 25–29⋅9 kg/m2, and obese: ≥30 kg/m2) were used to assess the BMI data. Diet component and energy intake data were obtained using the Diet History Questionnaire 1 (DHQ1) in the MGTT.,38 The DHQ1 was developed and validated by the National Institutes of Health (NIH) and completed by the participants of the MGTT at the baseline. Using data from the DHQ1, average food and nutrient intake was estimated using DietCalc software created by the National Cancer Institute. Physical activity was assessed by a health history questionnaire (HHQ) in the MGTT.,38 The HHQ asked participants questions about physical activity including the type, frequency, and duration of activity.,38 The average hours of each activity per week were multiplied by its metabolic equivalent (MET) hour equivalent to produce MET-hours for each activity for each participant weekly.,38 Age and antihypertensive medication use as well as vitamin and mineral supplement use were also obtained from the HHQ.,38Other variables used in this study were body mass index (BMI), waist-to-hip ratio (WHR), sodium, caffeine and alcohol consumption, physical activity, age, antihypertensive medication use, and vitamin and mineral supplement use. Values needed to calculate BMI and WHR were obtained by trained clinic staff in the MGTT. BMI numerical values and the corresponding CDC categories  and outcome variables (SBP and DBP). These analyses and clinical relevance were considered to identify potential confounding variables that would be covariates used in the adjusted models and included age, BMI, WHR, energy intake, energy, sodium, caffeine and alcohol consumption, physical activity, and antihypertensive medication use. Different linear regression models were constructed to explore associations between adiponectin, leptin, blood pressure measures, and COMT genotype. The models included the crude analyses with no adjustment, and models adjusted for age, BMI and physical activity as model 1; and model 2 with adjustment for age, BMI, WHR, physical activity, energy intake, sodium intake, caffeine intake, total drinks of alcohol per week, and adiponectin or leptin where appropriate. To explore the association between COMT genotype and blood pressure measures, a model was built and adjusted for age. In addition, we conducted subgroup analyses based on age, BMI category and blood pressure category, antihypertensive medication use and COMT genotype, adiponectin and leptin where appropriate. P-values for the interaction of age, BMI, COMT genotype, blood pressure category, current antihypertensive medication use, and adiponectin and leptin, where appropriate, on the blood pressure measures were calculated by a generalised linear model while also adjusting for age, BMI, and physical activity where appropriate.Descriptive statistics were reported for all variables. Histograms and boxplots were used to assess the normality of the data to identify any extreme outliers. Scatterplots were also used to visually assess the linearity of the relationships between the variables. Bivariate analyses were performed with ANOVA and Pearson's correlation to test for potential unadjusted relationships between predictor variables . P-values ≤0⋅05 were considered statistically significant.Since this was a secondary analysis of available data, a sd) age of the sample was 60⋅7 (5⋅0) years and the mean (sd) of BMI was 28⋅1 (2⋅9) kg/m2. The majority of participants were non-Hispanic (97⋅9 %), Caucasian (94⋅9 %), and had never smoked (67⋅9 %). More than 71 % of participants reported not using antihypertensive medications. Blood pressure categorisation revealed that 32⋅9 % of the sample population were categorised as normotensive, 20⋅7 % were categorised as having elevated blood pressure, and 46⋅4 % were categorised as hypertensive.Data were utilised for 237 participants from the MGTT for this study . The meaβ = 0⋅128, P = 0⋅049), however, the statistical significance was lost in adjusted analyses. Crude analyses also revealed that there was not a significant association between adiponectin with either SBP or DBP or between leptin and DBP. To further assess the association, two adjusted models were utilised, one in which BMI and physical activity were controlled for and another in which BMI, WHR, physical activity, energy intake, sodium intake, caffeine intake, total drinks of alcohol per week, and adiponectin or leptin where appropriate were controlled for. The results of these analyses remained unchanged from the crude analysis with the exception of leptin and SBP, as discussed.Associations between adiponectin, leptin, and blood pressure measures are displayed in COMT genotype, blood pressure category, and current blood pressure use with adiponectin and leptin and DBP. Tests of interaction revealed no significant interaction between adiponectin and leptin and SBP or DBP measures with age, BMI, COMT genotype, blood pressure category, and current antihypertensive medication use (see Supplementary Tables S1 and S2).Associations between adiponectin and leptin and blood pressure measures were explored further through stratified analyses exploring potential interactions of age, BMI, Associations between COMT genotype and blood pressure measures are also shown in COMT genotype and SBP or DBP measures with age, BMI, adiponectin, leptin, blood pressure category, and current antihypertensive medication use (see Supplementary Tables S3 and S4).Subgroup analyses revealed no significant interaction between COMT genotype and blood pressure measures in women who are post-menopause. While previous research has shown some evidence of such associations, this study found no association between blood levels of adiponectin and leptin or COMT genotype with blood pressure measures in women who were post-menopause.,19–22,34This study explored the potential associations between circulating adiponectin, leptin, and blood pressure measures and additionally examined the potential associations between ,16 A 5-year, prospective study including both men and women identified an inverse relationship between adiponectin and risk of hypertension; however, exploring these associations in women specifically is important as gender differences, with women usually having higher levels, have been reported.,16–18 Additionally, when adiponectin levels have been compared between women who are pre- and post-menopause, it appears menopause may contribute to further increases in circulating adiponectin levels, which may in theory may lead to lower blood pressures and potentially lower risk of hypertension. The findings of this study and other investigations which specifically studied women who are post-menopause have not supported the previously reported inverse relationship found in both men and women however.,18–22,43 Although not significant, analysis by July et al. revealed that women who were post-menopausal (mean age: 60⋅9 ± 9⋅4 years) and hypertensive had lower adiponectin levels compared to women who were normotensive. In the current study, even when data were stratified by blood pressure category, no association was found. Therefore, the results of the current study are consistent with previous research suggesting there is no association with either SBP or DBP values or blood pressure status (normotensive v. hypertensive) in this population.–22Mechanistically, adiponectin plays a role in endothelial function, nitric oxide production and supports the action of anti-inflammatory macrophages which ultimately may affect blood pressure.,24–28 Many studies have demonstrated a positive correlation between leptin and blood pressure.,29–32 This relationship has remained even among non-obese participants suggesting this relationship is independent of weight status.–32 Similar to adiponectin, women's leptin levels are generally higher than men's which is also independent of BMI. This gender difference has been associated with a larger proportion of fat mass in women compared to men relative to BMI. Menopause further increases leptin levels, which has been hypothesised to be related to an increase in adipose tissue. Although an association was not found in this study after adjusting for confounding variables, available research suggests there is a relationship between leptin and blood pressure in women who are post-menopause ranging from 50 to 68 years old primarily in relation to SBP.,22,34 For example, Olszanecka et al. reported a significant positive relationship between leptin and SBP. Furthermore, when analyses were conducted in consideration of blood pressure categorisation by Khokhar et al., leptin was strongly positively correlated with both systolic and diastolic blood pressures in women who were hypertensive and had a normal BMI. A similar pattern was observed for women who were hypertensive and obese. Using a multiple linear regression model, the findings of Khokhar et al. also support what is available elsewhere in the literature that a positive relationship exists between leptin and blood pressure, specifically SBP in those who are hypertensive, regardless of weight status. As noted, the associations identified by other researchers were not supported by the findings of this study. We identified a positive association between leptin and SBP in the crude analysis, however, the observed association was no longer statistically significant after adjustment for potential confounders or in stratified analyses.Leptin is another fat-derived hormone of particular interest in this study. Its primary role in the body is to be a messenger to the brain regarding available energy stores; however, leptin may also impact blood pressure status due to its effect on sympathetic nervous system activation at the renal level.COMT genotype.,36 Individuals possessing homozygous variant alleles (A/A) and heterozygous variant alleles (G/A) show reduced enzymatic activity compared to those with homozygous wild-type alleles (G/G) who have higher activity of the COMT enzyme.,9,35,36COMT genotype distribution varies based on ethnic backgrounds and the G/A variant is found in approximately 40 % of individuals.,38,44,45 In individuals of primarily Caucasian descent, the G/A variant is found in 40–51 % of the population.,44,45 In this study sample, the prevalence of the G/A genotype was 46 %. The A/A variant is the least common and is found in approximately 20 % of individuals or less.,38,44,45 Some studies using primarily Caucasian samples have reported a prevalence of this variant to be up to 21 %, however, the prevalence in this study sample was 31⋅6 %.,44,45 As COMT enzyme metabolises specific catecholamines, those with the low activity genotype have a lower capacity for catecholamine metabolism compared to the other genotypes; given the role that catecholamines play in blood pressure regulation a potential for increased blood pressure is plausible in this group.–9,36 A significant relationship between COMTA/A genotype and higher SBP and DBP was identified in a study completed in Japanese men as compared to those with the G/A or G/G genotypes. The A/A genotype was also associated with a higher risk of hypertension in the study completed by Htun et al. This study did not find a significant association between the COMT genotype and blood pressure measures; however, this finding may be due to differences in the sample population as participants in the present study were primarily Caucasian. In addition, previous studies have been completed inclusive of both male and female participants.,9 To the best of our knowledge, this is the first study exploring the association between COMT genotype and blood pressure measures in women who are post-menopause.COMT enzyme activity is dependent upon ,46 This study has some strengths including being adequately powered and it utilised a robust statistical approach that accounted for multiple potential confounding variables which have not been done consistently in previous research. As part of the research aim, this study also explored novel variables  in a specific population at increased risk of CVD which can also be counted as the strength of the present study.There are some limitations to the present study that are worth noting. The first limitation is that the study has a cross-sectional design, therefore, causality cannot be inferred. Secondly, this study is a secondary analysis of a large clinical trial, and the data were not prospectively collected with the aims of the current study in mind. As such the methods used to obtain the data in the MGTT may not have utilised the most ideal methods in consideration of the aims, such as that for blood pressure. Also, as a secondary analysis, this study is limited by the inclusion and exclusion criteria of the parent study. While data were available for many variables of interest within the dataset, we cannot rule out the possibility of residual confounding variables such as energy intake. Additionally, the inclusion and exclusion criteria of the parent study may be a limiting factor for the generalisability of this study as only women who were post-menopause and predominantly White from the Minneapolis-St. Paul metropolitan area were included in the MGTT.COMT genotype and blood pressure. This study utilised a more robust statistical model than much of the previous research has taken into consideration certain potential confounding variables . The data were also stratified by select participant characteristics to further explore patterns of change within the subgroups. Even with an adequately powered study and additional statistical analyses, this study did not identify significant associations between plasma leptin, plasma adiponectin, COMT genotype with blood pressure in women who are post-menopause. Future studies should use a more diverse population to better determine if race or ethnicity has any impact on the associations explored. In addition, this study only looked at baseline data from the MGTT which reflected one point in time, as with some existing research; future studies could utilise a prospective design to further explore the potential associations over time.Overall, this study adds to the available literature exploring the associations between circulating adiponectin and leptin, Green et al. supplementary material


Previous studies have found associations of red blood cell (RBC) traits (hemoglobin and RBC count) with blood pressure; whether these associations are causal is unknown.n = 167,785). Additionally, we performed bidirectional 2 sample Mendelian randomization (MR) analyses to explore the causal effect of the 2 traits on systolic (SBP) and diastolic blood pressure (DBP), using genetic instrumental variables regarding hemoglobin and RBC identified in UK Biobank  and International Consortium of Blood Pressure studies for SBP and DBP .We performed cross-sectional analyses in the Lifelines Cohort Study  and RBC . MR analyses suggested that higher hemoglobin and RBC cause higher DBP . Reverse MR analyses  suggested causal effects of DBP on both hemoglobin  and RBC . No significant effects on SBP were found.In cross-sectional analyses, we observed positive associations with hypertension and blood pressure for both hemoglobin (odds ratio [OR] = 1.18, 95% confidence interval [CI]: 1.16–1.20 for hypertension; Our results suggest bidirectional causal relationships of hemoglobin and RBC with DBP, but not with SBP.  In 2015, the number of deaths related to high blood pressure was more than 8 million, and the vast majority (88%) of them occurred in low- and middle-income countries.2 Modifiable risk factors for hypertension include low potassium intake, high sodium intake, overweight and obesity, unhealthy diet, lack of physical activity, cigarette smoking, and alcohol consumption.1 The pathogenesis underlying hypertension is complex and incompletely understood; further study into potential mechanisms is therefore warranted.Hypertension is an important cause of cardiovascular disease, premature death, chronic kidney disease, and dementia.3 Red blood cells are a primary determinant of blood viscosity, which has been suggested to be linked to blood pressure4 and cardiovascular events.5 Previous observational studies in different populations support such a link,6–12 whereas in trials in clinical samples , erythropoietin/erythropoiesis-stimulating agents caused elevated blood pressure.14 However, the observational studies are possibly affected by unmeasured confounding and reverse causation. Furthermore, generalizability of results from trials in clinical samples to the general population is uncertain. Thus, it remains unknown whether hemoglobin and RBC are causally related with blood pressure in the general population.Hemoglobin, the principal component of red blood cells (RBCs), performs an important function transporting oxygen and carbon dioxide into and out of the body’s circulation system.15Under the instrumental variable (IV) assumptions, the Mendelian randomization (MR) approach can overcome some of the problems of observational studies, such as confounding and reverse causation, thereby strengthening causal inference from observational data.16 (integrating results from different approaches with different key sources of bias to strengthen confidence in the findings)  summary data.19In the current study, we aimed to examine potential effects of 2 RBC traits  on blood pressure by applying 2 approaches within a triangulation frameworkindings) : first, 17 In brief, Lifelines is a multidisciplinary prospective population-based cohort study examining in a unique 3-generation design the health and health-related behaviors of 167,785 persons living in the North of the Netherlands. It employs a broad range of investigative procedures in assessing the biomedical, sociodemographic, behavioral, physical, and psychological factors which contribute to the health and disease of the general population, with a special focus on multimorbidity and complex genetics. The study adhered to the Declaration of Helsinki and was approved by the medical ethical committee (number 2007/152) of the University Medical Center Groningen, The Netherlands. Signed informed consent statements were obtained for all study participants prior to inclusion to the study. We created a directed acyclic graph (DAG) (http://dagitty.net/dags.html)20 to identify a minimally sufficient adjustment set (MSAS). This MSAS included age, sex, body mass index , education , nonoccupational physical activity , estimated glomerular filtration rate (estimated by CKD-EPI equation21), and smoking . We sampled adult subjects (≥18 years of age) from the Lifelines Cohort Study that had complete data on blood pressure , hemoglobin, RBC , and the variables in the MSAS. Hypertension was defined as systolic blood pressure (SBP) ≥140 mm Hg and/or diastolic blood pressure (DBP) ≥90 mm Hg or use of antihypertensive medication.23 In addition, we performed analysis using hypertension defined as SBP ≥130 mm Hg and/or DBP ≥80 mm Hg, as per the updated guidelines of the American College of Cardiology/American Heart Association (ACC/AHA).24 For those participants taking antihypertensive agents, SBP and DBP were adjusted by adding 15 and 10 mm Hg, respectively, to their observed values, which is a recommended approach to reduce bias and improve statistical power.25 Subjects  with missing data on hemoglobin, RBC, blood pressure and any of the covariates from the MSAS were removed, leaving 109,135 participants for the analysis   online uanalysis .stats and robustbase packages in R (version 4.0.3) were used. A 2-sided P < 0.0125  was considered statistically significant. We standardized our traits of interest  so that each had a mean of 0 and a variance of 1.Continuous data were represented as mean ± SD or median (interquartile range [IQR]), while categorical data were expressed by numbers with percentages. For each outcome , we constructed 3 models: model 1 was an unadjusted model; model 2 was adjusted for age and sex; model 3 was adjusted for the MSAS. Reverse analyses were performed using BP or hypertension as exposure, and hemoglobin and RBC as outcomes, while adjusting for the same set of variables. We applied a robust linear regression method as this method, compared with ordinary linear regression, is considered less sensitive to the wide distribution of blood pressure, especially potential outliers therein, we observed in our data see  online. 26  and International Consortium of Blood Pressure-GWAS 19 in this MR study. Details on these studies can be found in We used UK Biobank .18Blood samples were collected in 4 ml EDTA vacutainers for participants of the UK Biobank baseline cohort and analyzed at the UK Biobank central laboratory within 24 hours of blood draw. Measurement of hemoglobin and RBC was determined by Beckman Coulter LH750 Haematology Analyser  according to the standard process .28 Instrn = 757,601 Europeans, presented as “UKB-ICBP”), which is composed of UKB  and the ICBP-GWAS .19 For ease of comparison, we standardized blood pressure summary GWAS data using a Z-score transformation.We obtained summary data for the association of the hemoglobin and RBC-related variants with blood pressure from combined GWAS studies (27) for each included SNP was performed, which is a common approach to estimate the causal effect under the assumption that all SNPs are valid IVs.28 Several sensitivity analyses were conducted, i.e., heterogeneity assessment, leave-one-out MR analysis, outlier assessment, Mendelian Randomization-Egger (MR-Egger) regression,30 weighted median method,31 MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO),32 and Steiger filtering.33 We assessed heterogeneity by Cochran’s Q-statistic. We examined individual Wald estimates, leave-one-out analyses, and funnel plots to identify influential, potentially pleiotropic SNPs and overall directional pleiotropy.Random-effects inverse-variance weighted (IVW) meta-analysis of the Wald ratio  after Steiger filtering. To further investigate potential reverse causation, reverse MR analyses  were performed using genetic instruments for SBP and DBP.20 We therefore performed sensitivity analyses using summary data on blood pressure from UKB (presented as “UKB only”) that were not adjusted for BMI. However, this resulted in complete sample overlap which may exacerbate weak instrument bias and bias toward the observational estimate.15 We therefore performed additional sensitivity analyses using summary data from ICBP . In addition, we applied MVMR including BMI as a covariate as this has recently been shown to counter potential collider bias due to BMI adjustment.34 Here, we did not apply Steiger filtering,33 as it is not well established in the MVMR framework. Due to overlap of SNPs between hemoglobin and RBC, as well as between SBP and DBP, there is potential violation of the exclusion restriction assumption through horizontal pleiotropy (https://gwas.mrcieu.ac.uk/)35 to identify potential pleiotropic variants genome-wide significantly (P < 5 × 10−8) associated with any of the a priori defined confounders  were used to perform this MR analysis. A 2-sided threshold of P < 0.0125  was considered statistically significant. This MR study followed the STROBE-MR Checklist of Recommended Items to Address in Reports of MR Studies /l, respectively. Medians of SBP and DBP were 125 (IQR 115–137) mm Hg and 74 (IQR 68–81) mm Hg, respectively. Prevalence of hypertension in this population was 26.1%    years. Mean ± SD of hemoglobin and RBC were 14.11 ± 1.27 g/dl and 4.71 ± 0.40   online. 152,728)  online, 152,728)  online.B = 0.11 SD per SD higher hemoglobin, 95% CI: 0.11–0.12), and DBP  in the MSAS-adjusted models. Furthermore, RBC was also positively associated with hypertension , SBP , and DBP  in the MSAS-adjusted models , SBP  are shown in F parameter for the 2 traits and found sufficient instrument strength (91.1 for hemoglobin and 107.1 for RBC). All individual SNPs had an F > 10, which is further evidence of sufficient instrument strength.We calculated a 1-sided lower-bound confidence limit of the B = 0.00 SD per SD higher hemoglobin, 95% CI: −0.04 to 0.04) or RBC on SBP   and RBC on DBP . MR sensitivity analyses yielded consistent results . RBC was positively associated with DBP .We found no significant effects of hemoglobin on SBP (IVW to 0.05)  online.  results  online. P value = 0.527) and the RBC–DBP analysis . The estimates of MVMR and IVW models from UKB-ICBP, UKB only and ICBP only datasets were again consistent, suggesting that our results were unlikely to be severely affected by collider bias  and 1.3 mm Hg (95% CI: 1.1–1.4) per mmol/l higher hemoglobin for women and men, respectively, based on a large sample of more than 100,000 Dutch voluntary blood donors, and similar results were found for DBP ,6 which supports our findings. Interestingly, a large randomized, double-blind, placebo-controlled trial  showed an increase in DBP  after administration of darbepoetin alfa, an erythropoiesis-stimulating agent. Concordant with our study, SBP did not increase.13 In an open-label trial involving 1,432 patients with chronic kidney disease in the United States, DBP increased by 0.2 ± 12.9 mm Hg in high-hemoglobin group and decreased by 0.7 ± 12.4 mm Hg in low-hemoglobin group, whereas SBP decreased in the both groups compared with baseline levels, although the impact of antihypertensive drugs cannot be ruled out.14 Discordant with our study, a large MR study  using a strong functionally validated instrument suggested that higher endogenous erythropoietin levels causes lower DBP , but not lower SBP .38 A potential explanation for this discordant effect is that endogenous erythropoietin levels may also be higher due to potential compensatory mechanisms in response to proneness to anemia; endogenous erythropoietin, as an upstream proxy of RBC and hemoglobin, may thus potentially show opposite effects to these traits. In a 24-week, multicenter trial from 15 countries (n = 252 chronic kidney disease patients), daprodustat  and controls (received recombinant human erythropoietin) did not markedly impact SBP and DBP. The authors propose that this was related to smaller drug doses and/or adequate control of blood pressure.39 None of the abovementioned studies examined a potential reverse causal relation between hemoglobin/RBC and DBP. In summary, our results are generally consistent with previous studies with large sample size and suggest that hemoglobin and RBC contribute to a higher DBP and the development of hypertension, although some inconsistencies remain that warrant follow-up study.Our observational results are consistent with previous studies.40 which has an antihypertensive effect.41 Not only that, arginase released by hemolysis can retard NO generation.40 Moreover, free hemoglobin, even when present at low levels, released by damaged RBCs could not only play a role in scavenging NO activity through dioxygenation reactions, but also induce vasoconstriction, inflammation, and oxidative stress, a state with greater amount of reactive oxygen species than the normal physiological range.43 The generation of these reactive oxygen species might also suppress the bioactivity of NO.44 In our study, we only found associations of hemoglobin and RBC with DBP, but not with SBP. One possible explanation is that the abovementioned microparticles, arginase and free hemoglobin from RBCs have the function of scavenging NO activity.40 While blood flow velocity around the center of the vessel is higher than that near the walls of blood vessels attributed to the friction force. This forms a pressure difference that drives RBCs toward the center of the vessel far away from the NO synthesizing endothelium.40 Blood flow velocity is not constant in common carotid artery, it is faster during systole than that during diastole.45 When the flow velocity is decreased (corresponding to DBP), the endothelium may be more susceptible to exposure to RBCs and its related substances, such as free hemoglobin, compared with the higher flow velocity situation during SBP, thus inactivating NO and inducing higher DBP. In MR, we found a reverse effect of blood pressure on RBC traits, which we did not anticipate. We are unaware of previous literature describing such an effect, and more research is needed to validate this finding and elucidate the mechanisms (if any) underlying this relation. In exploratory analyses, we in addition found evidence for a bidirectional relation between hematocrit and DBP. We found no evidence for a relation of RBC distribution width, mean corpuscular volume, mean corpuscular hemoglobin concentration, or mean corpuscular hemoglobin, with blood pressure. This supports a role for RBC count and total hemoglobin, rather than RBC volume or corpuscular hemoglobin. Furthermore, it lends support for previous observations that polycythemia is  related to hypertension,46–48 given that even with physiologically higher RBC counts and hemoglobin concentrations, effects on blood pressure are present.We found evidence for causal effects of hemoglobin/RBC on DBP. A possible explanation is that microparticles released from RBCs could exert its function in inactivation of nitric oxide (NO),15 is the greatest threat to MR. We therefore performed several sensitivity analyses that allow for a range of violations of this criterion. These analyses yielded consistent results and therefore we deem risk of bias due to horizontal pleiotropy small.MR studies provide unconfounded estimates for an exposure–outcome pair under the IV assumptions discussed in the Methods section. Arguably, horizontal pleiotropy of instrumental SNPs, which is a violation of the exclusion restriction criterion,This is the largest study to date that investigated the associations between the RBC traits, hemoglobin and RBC, and blood pressure. Our observational analysis utilizes extensive information on covariates, allowing us to explore the optimal set of confounders. An additional advantage of our analyses is that the complementary design  increases the reliability of these findings.Our study also has some limitations. First, high-level evidence to inform the DAG, such as prospective and experimental studies, was not always available. This may have led to misoriented arrows in the DAG, and as a result, suboptimal covariate adjustment. Second, at the time of analysis, we only had data on medication use for baseline and not for follow-up; this precluded meaningful longitudinal observational analyses. Third, MR is thought to estimate a lifetime effect of an exposure on an outcome; short-term effects or potential treatment effects may be smaller. Fourth, our study was done in samples of the general population of European ancestry; generalization to the clinic and other ethnicities is therefore uncertain. Fifth, in MR, associations can be interpreted as causal effects, but only if the MR assumptions (some of which untestable) hold. Functional work is required to definitively establish causality.Our findings suggest a modest causal effect of RBC traits on DBP, providing insights into physiology. Future study could assess whether intervention on RBC traits would translate into meaningful protection against hypertension and sequelae thereof, and whether it is safe to do so given the risk of anemia. If not suitable as a drug target, RBC traits could instead play a role in risk stratification; future study could assess whether inclusion of RBC traits, on top of traditional risk factors, improves prediction of hypertension. Additionally, future work may involve investigating potential sex differences in the relation between hemoglobin and RBC with blood pressure, using sex-stratified GWAS data.In conclusion, our results suggest bidirectional causal relationships between hemoglobin, RBC, and DBP, but no causal relationships with SBP.hpad061_suppl_Supplementary_MaterialClick here for additional data file.hpad061_suppl_Supplementary_TablesClick here for additional data file.


This study aimed to evaluate the effects of an 8-week self-administered moxibustion program on blood pressure, stress, sleep quality, and quality of life in hypertensive patients. We compared its effects with those of self-acupressure to assess the feasibility of using moxibustion as a self-management method for hypertension. Forty-six subjects were recruited and randomly assigned to a moxibustion group and an acupressure group. The moxibustion group applied moxa sticks to seven acupoints by themselves five times a week for 8 weeks, while the acupressure group placed acupressure patches on the same acupoints by themselves. Systolic and diastolic blood pressures were measured. Stress was assessed with plasma epinephrine, plasma norepinephrine, and the Perceived Stress Scale (PSS). Sleep quality and quality of life were evaluated using the Pittsburgh Sleep Quality Index (PSQI) and the World Health Organization Quality of Life-Brief Version (WHOQOL-BREF), respectively. Systolic and diastolic blood pressures decreased in the moxibustion and acupressure groups to a similar extent after the 8-week intervention. Plasma epinephrine and norepinephrine levels and PSQI variables were not changed in both groups. PSS decreased only in the moxibustion group. Quality of life related to physical health and environmental health, as assessed by the WHOQOL-BREF, significantly improved to a similar degree in both groups. Both self-administered moxibustion and acupressure therapies were effective in reducing blood pressure in hypertensive patients. Hypertension, defined as persistently high blood pressure, is the major cause of heart disease, stroke, and all-cause death globally ,2. Most Although antihypertensive medications are effective in lowering blood pressure, some patients fail to adhere to medication and have uncontrolled blood pressure with medication, in some cases due to resistant hypertension, which poorly responds to medication ,5. Due tHypertension is a common chronic disease that requires continuous treatment and management . Self-caAmong non-pharmacological treatments, acupoint stimulation therapies such as acupuncture, moxibustion, and acupressure have shown effectiveness in reducing blood pressure ,10,11,12Taken together, moxibustion has demonstrated its effectiveness in lowering blood pressure ,16. MoreIn addition to managing blood pressure, it is important to consider quality of life in hypertensive patients, as they often experience a low quality of life compared to individuals with normal blood pressure . HoweverStress and poor sleep quality are well-known risk factors for hypertension and can contribute to a lower quality of life in hypertensive patients ,19,20. HPsychological stress is known to increase sympathetic nerve activity and stress hormone levels , which can lead to elevated blood pressure . HoweverAccordingly, the purpose of this study was to verify the effectiveness of an 8-week self-administered moxibustion intervention in decreasing blood pressure, improving sleep quality and quality of life, and reducing perceived stress and epinephrine and norepinephrine levels in hypertensive patients. Additionally, we aimed to investigate whether self-moxibustion has greater effects on these outcomes compared to self-acupressure.This study was conducted as a parallel randomized clinical trial from January to June 2022. This study was approved by the Institutional Review Board of Kyonggi University (KGU-20210527-HR-070-02). Prior to participating in this study, all participants provided written informed consent. This study was registered on CRIS.nih.go.kr under the Korean Clinical Trial Registry (KCT0007424). Furthermore, this study adhered to the guidelines of the Consolidated Standards of Reporting Trials (CONSORT) .n = 23) or the acupressure group (n = 23). Each participant selected a sealed and opaque envelope from a box, and their group assignment was determined by the labeled paper inside the envelope.Subjects were recruited from the primary care clinic and continuing education center for moxibustion and meridian studies in Seoul, South Korea. A total of 52 participants were initially screened for eligibility based on inclusion and exclusion criteria. The CONSORT flowchart of this study is presented in After the group assignment, two subjects withdrew from this study due to a change of mind. Therefore, a total of 22 subjects in each group underwent the intervention. Three subjects dropped out due to conflicts with their schedules. Finally, 21 subjects in the moxibustion group and 20 subjects in the acupressure group completed this study.Each subject in the respective assigned group mastered the acupressure or moxibustion technique and underwent a consistency check to ensure proficiency. Adherence to antihypertensive medication and daily self-administration logs were recorded throughout the study. Systolic and diastolic blood pressures, perceived stress, sleep quality, and quality of life were measured at baseline and after 8 weeks. Additionally, blood samples were collected at baseline and after the 8-week intervention to evaluate levels of epinephrine and norepinephrine and a complete blood count.The inclusion criteria for this study were as follows: men and women aged 19 years or older; diagnosed primary hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg); currently taking antihypertensive medication; or having prehypertension (systolic blood pressure between 130 and 139 mmHg or diastolic blood pressure between 85 and 89 mmHg) . The excSubjects in the moxibustion group self-administered the treatment by applying 5 moxa cones  a directlSubjects in the acupressure group self-applied acupressure patches, which had a metal stud pressing down on the acupoints b, on theThe measurements included blood pressure, levels of epinephrine and norepinephrine, a complete blood count, perceived stress levels, sleep quality, quality of life, and adverse events. The resting systolic and diastolic blood pressures were measured in the morning using the auscultation method with a mercury sphygmomanometer and a stethoscope . Before the measurement, each subject was comfortably seated and allowed to relax quietly for 10 min. Blood pressure was then measured with the subject in a seated position, with their arm resting on a table and positioned at the level of the heart. The cuff was placed on the bare upper arm and adjusted according to the individual’s arm circumference.The blood samples were sent to a commercial laboratory  for a complete blood count and catecholamine analysis. Plasma samples were prepared by centrifuging the blood samples at 3000 rpm for 10 min, and they were collected using anticoagulant (EDTA)-treated tubes. The plasma epinephrine (pg/mL) and norepinephrine (pg/mL) levels were measured using high-performance liquid chromatography with electrochemical detection. This analysis was conducted using plasma catecholamine reagent kits  and an electron capture detector .The subjective stress level was evaluated using the Korean version of the Perceived Stress Scale (PSS), which consists of 10 items . Each itSleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI), which measures subjective sleep quality over the past month . The totQuality of life was evaluated using the Korean brief version of the World Health Organization Quality of Life (WHOQOL-BREF) questionnaire . The WHOAdverse events related to the intervention were recorded. Furthermore, the complete blood count analysis included measurements of red blood cells, white blood cells, platelets, hemoglobin, and hematocrit (%).We planned to utilize a two-way analysis of variance (ANOVA) with repeated measures to examine the significance of intergroup differences in changes from baseline. Sample size calculation was performed using the G*Power program, version 3.1.9.4, to detect the significant interaction of the 2-way ANOVA with repeated measures. For a medium effect size  , assuminThe data were analyzed using SPSS version 27.0 . A significance level of 0.05 was set for all analyses. The normality of the distribution of continuous data was assessed using the Shapiro–Wilk test. In cases where the data were found to not follow a normal distribution, non-parametric statistics were used to determine statistical significance. Chi-square tests were used for categorical variables, while Mann–Whitney tests were conducted for age and body mass index to evaluate the significance of intergroup differences at baseline.If all four data sets (2 × 2: data from moxibustion and acupressure groups at baseline and week 8) exhibited a normal distribution, a two-way ANOVA with repeated measures was conducted to assess the significance of intergroup differences in changes from baseline (group-by-time interaction). The factors included in the main effect were time (baseline vs. week 8) and group (moxibustion vs. acupressure). Two-way ANOVA with repeated measures was conducted on the PSQI total score, the three domains of WHOQOL-BREF , and the hematocrit. Results of some variables, including PSQI, domains of WHOQOL-BREF, and the blood cell count, that were analyzed using non-parametric statistical analyses are presented separately in the tables.t-tests or Wilcoxon signed-rank tests. To evaluate the significance of intergroup differences, independent t-tests or Mann–Whitney tests were performed.We conducted non-parametric statistical analyses on systolic and diastolic blood pressures, epinephrine, norepinephrine, PSS, the PSQI subscales, the WHOQOL-BREF social relationship domain, and four variables of blood cell counts. In cases where the data did not exhibit a normal distribution, the significance of intragroup differences was tested using paired 2) for two-way ANOVA with repeated measures; Cohen’s d for t-tests; and the r-values for the Wilcoxon signed-rank test and Mann–Whitney test [2 = 0.01; d = 0.2), medium , and large  effects for partial eta squared and Cohen’s d [The Pearson correlation coefficient (r) was used to assess the degree of linear association between baseline blood pressure and the magnitude of blood pressure change (week 8–baseline). Additionally, effect size estimates of the statistics were calculated using the following formulas: Partial eta squared , 17 subjects in the moxibustion group and 18 subjects in the acupressure group accomplished this requirement. The average completion rate of the sessions was 97.5% in the moxibustion group and 97.3% in the acupressure group.2 with a standard deviation of 3.7 kg/m2. There were no significant intergroup differences in gender proportion or age. However, the acupressure group had a significantly higher body mass index compared to the moxibustion group (p < 0.05). Most subjects (82.9%) had a college education or higher. Approximately 95% of the subjects were married, and the majority (95%) were non-smokers. Furthermore, 46.3% of subjects reported being non-drinkers, and 85.4% did not engage in moderate-to-vigorous-intensity exercise. There were no significant intergroup differences in terms of education, marital status, exercise, smoking, or drinking.The subject characteristics are summarized in n = 14) had uncontrolled hypertension, defined as systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg, with medication (n = 7) or without medication (n = 7). In the acupressure group, 90% of subjects (n = 18) had uncontrolled hypertension, with medication (n = 10) or without medication (n = 8).Among the medications taken by the subjects, the majority were for managing conditions such as hypertension, diabetes mellitus, and hyperlipidemia. In the moxibustion group, two subjects had prehypertension. Among the 39 hypertensive subjects, 24 individuals were taking antihypertensive medication. Approximately 74% of hypertensive subjects in the moxibustion group  and the acupressure group (p < 0.05) after the 8-week intervention. Similarly, diastolic blood pressure significantly decreased in both the moxibustion group (p < 0.01) and the acupressure group (p < 0.01). Results from the Mann–Whitney U test and independent t-test indicated that there were no significant intergroup differences in the extent of changes from baseline in systolic and diastolic blood pressures (As shown in ressures .n = 41) revealed that the magnitude of reduction in blood pressure was significantly correlated with baseline blood pressure  and diastolic blood pressure , indicating that subjects with higher baseline blood pressures experienced a greater blood pressure reduction with the treatments.Correlation analysis of pooled data from two groups (p < 0.05). Nonetheless, there were no significant intergroup differences in the extent of change from baseline for PSS  and environment domain (p < 0.05) of the WHOQOL-BREF in both groups, indicating a significant main effect of time without the presence of an interaction effect.p < 0.05) in the moxibustion group. However, no significant improvement was observed in the acupressure group. Nevertheless, the intergroup difference in the change from baseline did not reach statistical significance based on the Mann–Whitney U test.As presented in p-values and mean values compared to those in the per-protocol analysis. However, the findings from the intention-to-treat analysis remained consistent with those of the per-protocol analysis.In the intention-to-treat analysis for all PSQI and WHOQOL-BREF variables, there were slight changes in the n = 1) in the moxibustion group and skin redness (n = 1) in the acupressure group. In both the per-protocol analysis (Reported adverse events included blister occurrences (analysis  and the This study aimed to evaluate the effects of 8-week moxibustion self-care on blood pressure, stress, sleep quality, and quality of life in hypertensive adults, comparing its effects with those of acupressure self-care. The main finding of this study was that both moxibustion and acupressure self-care were effective in reducing blood pressure and improving quality of life, specifically in relation to physical health and the environment. Interestingly, perceived stress levels and quality of life related to social relationships improved only with moxibustion self-care. However, stress hormones, including epinephrine and norepinephrine, as well as sleep quality, did not show significant changes after the intervention.Moxibustion has been commonly used as a self-care practice at home in Asian countries for various health conditions ,38. HoweAcupressure is a non-invasive treatment option that can be easily administered by patients themselves via patches and bands . PreviouOur results demonstrated that both self-administered moxibustion and acupressure therapies led to a similar reduction in systolic and diastolic blood pressures. We initially speculated that moxibustion might have a greater effect on blood pressure compared to acupressure due to its additional merits. Moxibustion involves the use of heat and aroma, which are not present in acupressure . These eThere is an issue that the pre-treatment blood pressure levels have to be taken into account when comparing the efficacy of different antihypertensive treatments . The pheMany hypertensive patients are not receiving treatment, and a significant number of patients have uncontrolled high blood pressure exceeding 140/90 mmHg ,6. AchieIn our study, subjects easily learned and applied the techniques of moxibustion and acupressure after a short training course. The dropout rate was low, and overall session adherence at home was excellent. Our results indicate that both therapies can be effective self-care methods for lowering blood pressure with satisfactory adherence. Taking all these aspects into consideration, self-care practices involving moxibustion and acupressure can serve as additional treatment options for blood pressure control in pre-hypertensive and hypertensive patients, complementing conventional treatments.Previous research has implicated stress as a risk factor for hypertension ,19,20. HThe effect of moxibustion on stress, including stress hormones and sympathetic activity, has primarily been investigated in stress-induced animal models ,24. In tThe effect of moxibustion on sleep in hypertensive patients is not well understood. In our study, we observed that the total and subscale scores of the PSQI did not significantly change in both groups after the intervention. Similarly, Shin et al. also reported no effects of a 4-week moxibustion therapy on the total score of the PSQI in prehypertensive and hypertensive patients, although they did not provide the results for the PSQI subscales .The results of our study demonstrated that both the moxibustion and acupressure groups experienced improvements in the domains of physical health and environment, whereas social relationships showed improvement only in the moxibustion group. In contrast, Shin et al.  did not This study has several limitations. It should be acknowledged that some subjects may not have strictly adhered to the protocol guidelines for moxibustion and acupressure due to the nature of self-administration. This potential non-compliance could have influenced the study’s results. Thus, additional research is warranted to compare the effects of self-administered moxibustion and acupressure with those performed by practitioners. Nonetheless, this study represents the first investigation assessing the feasibility of moxibustion and acupressure as self-management methods for hypertension. Additionally, our findings provide the first evidence suggesting that moxibustion therapy may have potential clinical benefits for reducing stress.This study demonstrated that self-administered moxibustion and acupressure therapies resulted in a significant decrease in systolic and diastolic blood pressures among hypertensive patients, with no significant difference between the two. The reported adverse events were minor, and the participants showed good adherence to the therapies. These findings indicate that both moxibustion and acupressure can be effective self-management methods for reducing blood pressure in hypertensive patients. Additionally, improvements in quality of life, specifically related to physical health and the environment, were observed in both therapy groups. It is noteworthy that only moxibustion showed improvements in perceived stress levels and quality of life related to social relationships; however, further studies are needed to support these results. Overall, these findings suggest that both moxibustion and acupressure therapies hold promise as self-care methods for the prevention and management of hypertension and its associated symptoms.


Transcutaneous oxygen pressure (TcPO2) is used to assess microcirculation clinically; however, it is not widely available especially in rural hospital. The study was designed to explore potential alternatively biomarkers to assess microcirculation in diabetic kidney disease (DKD).A total of 404 patients from Xuzhou first hospital were recruited according to the case records system. Patients were grouped via the ratio of albuminuria and creatinine . Biomarkers in different ACR groups were compared by analysis of variance. Correlation analysis was determined by Pearson or Spearman analysis and binary logistic regression. The receiver operating characteristics (ROC) curve was performed to elucidate the prediction effect of ACR on TcPO2.p < .05). Frequency of low TcPO2 (<40 mm Hg) was markedly increased as increment of ACR stages with 30.2% in the ACR3 group (p < .01). There was a negative correlation between TcPO2 and age, ACR, chronic kidney disease (CKD), fast blood glucose, diabetes mellitus (DM) duration, and diabetic neuropathy. Further, binary logistic regression showed ACR was an independent influence factor for low TcPO2. After adjusting for age, gender, hypertension, DM duration, body mass index, glycated hemoglobin, diabetic neuropathy, and CKD, ACR was still an independent influence factor for TcPO2 . The area under the ROC curve was 0.768  for ACR. The analysis of ROC curves revealed a best cutoff for ACR was 75.25 mg/g and yielded a sensitivity of 71.7% and a specificity of 71.7%.A total of 404 diabetic patients were recruited with 248 patients diagnosed as DKD and 156 non‐DKDs. Age and cystatin C were significantly higher in the ACR3 group compared with those in the ACR1 group, whereas glomerular filtration rate, low‐density lipoprotein cholesterol, and TcPO2 were markedly decreased in the ACR3 group  could be used to assess microcirculation, which revealed endothelial injury. As endothelial injury is a cause of albuminuria in diabetic kidney disease (DKD), albuminuria and creatinine (ACR) might be employed to assess microcirculation in DKD patients in rural hospital where TcPO2 is not available. Our study showed TcPO2 was significantly lower in patients with ACR >300 mg/g and a negative correlation between TcPO2 and ACR was observed. Further, ACR was an independent influence factor of low TcPO2. On top of that, the area under receiver operating characteristics curve was 0.768 for ACR in predicting TcPO2. The best cutoff value for ACR was 75.25 mg/g and yielded a sensitivity of 71.7% and a specificity of 71.7%. These results suggested ACR could be used as a potential biomarker for assessing microcirculation in DKD patients. TcPO2 <25 mm Hg could be used to predict increased 1‐year mortality in diabetic foot ulcer patients.Previous study also proved TcPO2 could predict diabetic complications, including diabetic kidney disease (DKD). However, no data to date are available about the correlation of TcPO2 and DKD biomarkers. As is known, DKD is also associated with disorders of microcirculation, which is essential to be assessed in DKD patients. Nevertheless, TcPO2 is not widely used, especially in rural hospitals. It is important to explore alternative biomarkers to assess microcirculation in DKD.2A total of 404 patients were recruited from January 2018 to August 2019 in Xuzhou First People's Hospital. We excluded subjects as follows: younger than 18 years old, heart failure, fever, liver dysfunction, pregnancy, nonketotic hyperosmolar coma, diabetic lactic acidosis, diabetic ketoacidosis, and acute kidney injury.2.1Cystatin C, serum creatinine (sCr), uric acid, β2microglobin, serum albumin, calcium, phosphorus, serum lipid, and proteinuria were determined by Beckmann AU5800 automatic biochemical analyzer. Ratio of albuminuria and creatinine (ACR) was determined via immunoturbidimetry. Glycated hemoglobin (HbA1c) was determined by HPLC (BIO‐RAD). Homocysteine (HCY)was detected by enzymatic cycling assay.2.22; stage 2, 60 mL/min/1.73m2 ≤ eGFR <90 mL/min/1.73m2; stage 3, 30 mL/min/1.73m2 ≤ eGFR <60 mL/min/1.73m2; stage 4, 15 mL/min/1.73m2 ≤ eGFR <30 mL/min/1.73m2; stage 5, and eGFR <15 mL/min/1.73m2. Chronic Kidney Disease Epidemiology Collaboration (CKDEPI) fourlevel race equation was employed to calculate eGFR. The specific CKDEPI fourlevel race GFR estimation equation was as follows,Different CKD stages were divided according to estimated glomerular filtration rate (eGFR) following Kidney Disease: Improving Global Outcomes (KDIGO) guideline2.3ACR groups were defined as follows: ACR1 group, ACR <30 mg/g; ACR2 group, 30 mg/g ≤ ACR ≤ 300 mg/g; and ACR3 group, ACR > 300 mg/g.2.4DPN was diagnosed according to the American Diabetes Association guideline of 2010.2.4.1TcPO2 measurements were performed at the dorsum of both feet. The patients position was supine. The determination of TcPO2 should not be performed in edema feet. If the patients manifested edema feet, edema should be improved before determination.2.5p value <.05 was considered to be statistically significant.Statistical analysis was performed by PASW 22.0 statistical software . Data were expressed as mean ± SD. One‐way analysis of variance was used to compare means for continuous variables. The least significant difference method was used for continuous variables with homogeneous variances and Dunnett's method was used for continuous data with uneven variances. The Pearson or Spearman correlation analysis was employed to determine the correlations between TcPO2 and other indices. In addition, the binary logistic regression was employed to explore independent influence factors for TcPO2. Receiver operating characteristic (ROC) curve was built to evaluate the predicting ability of ACR on TcPO2. The best cutoff for the ROC curve was calculated with the Youden's index. 33.1p <.05). A lower level of hemoglobin was revealed in the ACR3 group (129.74 ± 18.0 g/L) compared with that in the ACR1 and ACR2 groups (p < .05) whereas no significant difference was observed between the ACR1 (139.16 ± 16.1 g/L) and ACR2 groups (135.12 ± 16.0) (p > .05). Levels of albumin were decreased as increments of ACR levels with the lowest in the ACR3 group (37.56 ± 4.84 g/L). Diabetes mellitus (DM) duration in the ACR2 and ACR3 groups were remarkably higher than that in the ACR1 group. No significant differences were observed in gender, body mass index (BMI), HbA1c, uric acid, serum creatinine, total cholesterol, triglyceride, high density lipoprotein, HCY, and C‐peptide among different groups , and TcPO2 were markedly decreased in ACR3 group (p < .01)  was markedly increased as increment of ACR stages with 30.2% in the ACR3 group     for ACR. The analysis of ROC curves revealed a best cutoff for ACR was 75.27 mg/g and yielded a sensitivity of 71.7% and a specificity of 71.7% . The area under the ROC curve was 0.768 . The funders had no role in study design, data collection and analysis, decision to publish, or reparation of the manuscript.The authors have nothing to disclose.