Diabetic nephropathy (DN) seems to be the major cause of chronic kidney disease that may finally lead to End Stage Renal Disease. So, renal function assessment in type 2 diabetes mellitus (T2DM) individuals is very important. Clearly, DN pathogenesis is multifactorial and different proteins, genes and environmental factors can contribute to the onset of the disease. We assessed sensitive and specific biomarkers (in blood and urine) which can predict kidney disease susceptibility among T2DM patients. Serum cystatin-c (cyst-c) in blood and urinary hemeoxygenase (HO-1) in addition to ACE I/D polymorphism and ACE G2350A genotypes. Hundred and eight T2DM patients and 85 controls were enrolled. Serum cystatin-c and urinary (HO-1) were tested by ELISA. Genetic determination of both ACE I/D polymorphism and ACE G2350A genotypes was performed by PCR for all participants. Significant rise in serum cystatin-c and urinary HO-1 levels were shown in diabetic groups compared with control group. Moreover, GG genotype of ACE G2350A gene in diabetic group was associated with rise in serum cystatin-c and urinary HO-1 compared with control group. Mutant AA genotype demonstrated increase in urinary HO-1. DD polymorphism was associated with rise in serum creatinine and cyst-c in diabetic group. Positive correlation was seen between duration of diabetes and serum cyst-c and between serum glucose and urinary (HO-1) in diabetic group. The results from this study indicated an association of serum cystatin-c with GG genotype of ACE G2350A in conjugation with DD polymorphism of ACE I/D which could be an early predictor of tubular injury in T2DM diabetic patients. In T2 DM, diabetic nephropathy (DN), where proteinuria is the main characteristic, represents one of the most frequent microvascular complication2. As early intervention, diagnostic markers could detect DN at early stage. It can slow kidney function deterioration and aid in reducing adverse outcomes. Pathologically, kidney hypertrophy is usually shown in DN patients with basement membrane thickening, extracellular matrix proteins deposition, glomerular sclerosis and interstitial fibrosis3. Although follow up of DN patients can be done by blood glucose and blood pressure control, progression of renal failure can eventually be seen in many patients4. Hence, understanding DN pathophysiology and development of new biomarkers seem to be of high demand for DN early diagnosis.Diabetes mellitus (DM) represents one of the most common chronic metabolic diseases with increasing prevalence5. It is synthesized by all nucleated cells at a stable rate and different factors such as sex, protein ingestion, inflammation, or muscle mass had no impact on its concentration6. It represents an ideal marker of endogenous glomerular filtration rate. For assessment of renal function, cystatin-c represents a more sensitive parameter than serum creatinine5. In critically ill adults, it is associated with recovery prognosis of acute kidney injury7. In addition, Taha et al.8 deduced that, in cadmium nephrotoxicity, cystatin-c is the most applicable biomarker.Cystatin-c, , is an endogenous cysteine protease inhibitor that is produced in most tissues. It is found also in all biological fluids10. Under homeostatic conditions, low levels of HO-1 protein in found in the tubules. While under several stress conditions that include oxidative stress, heat shock, hypoxia, heavy metals and toxins, heterogenous expression of HO-1 is detected in different renal compartments. In the medulla HO-1 is more highly expressed than the cortex, while in tubules, its expression is found to be very strong . In contrary to glomeruli where minimal or absence in HO-1 expression11. At present, in DN, assessment of urinary heme oxygenase-1 (uHO-1) was close to tubular damage12. Upregulation of HO-1 in proximal tubule cells seems to be an efficient biomarker of intrarenal activity as well as renal injury13. Recent study showed that, in patients with various kidney diseases, HO-1 levels within damaged cells falls away into the renal tubules14.Heme oxygenase 1 (HO-1) is known as an inducible enzyme which has different properties e.g., potent antioxidant, anti-inflammatory and anti-apoptotic one. Its activity was shown to occur via breakdown of heme fraction and generate protective products like carbon monoxide (CO) and biliverdin, with subsequent formation of bilirubin and ferritin via iron release from the heme part15 reported that ACE gene is located on chromosome 17q23 and spans approximately 21 kb. This gene was shown to include 26 exons as well as 25 introns and encodes a protein containing 1,306 amino acid. Previous records of National Center for Biotechnology Information (NCBI) had shown that ACE gene had more than 160 polymorphisms (mainly single nucleotide polymorphisms (SNP)16. Within the circulation, ACE G2350A gene (one of the ACE genes) had demonstrated a significant role in the determination of ACE level8.Angiotensin I-converting enzyme (ACE) is a key component of renin–angiotensin system (RAS). Its synthesis was observed in both epithelial and endothelial cells that is located in different organs as lungs, kidneys and blood vessels. Its main function is to catalyze producing vasoactive peptide angiotensin II from angiotensin I (its precursor), in addition to inactivate bradykinin. Besides, angiotensin II has a critical role in maintaining blood pressure, sodium homeostasis and renal hemodynamics. Mahwish et al.16. This produces three genotypes 18. It was recorded in individuals with DD genotype, higher concentration of ACE in both tissue and plasma ACE in comparison with either ID of II genotypes20.Other polymorphism is the insertion/deletion (I/D) one which represents the most widely studied ACE polymorphism. It is characterized by the presence of a 287 bp Alu repeat sequence (insertion) or absence (deletion) in intron18 revealed that ACE insertion (I)/deletion (D) polymorphism could cause instability of RAS as well as dysregulation of micro and macrocirculation and electrolytic homeostasis of the kidney. Moreover, it could affect or lead to DN through a direct effect on cellular hypertrophy resulting vascular modulation in the kidney.Silveira et al.Our goal is to investigate and assess serum cystatin-C and urinary HO-1 in the early diagnosis of renal injury  and its association with ACE I/D and G2350A genotypes in T2DM patients.Table Table p < 0.003) and significant distribution in ACE I/D gene (p < 0.049) in diabetic patient compared with control group.Table 1C (%) and serum glucose in all genotypes of ACE G2350A gene. Also, GG genotype of ACE G2350A gene in diabetic patient group was associated with significant increase in levels of both serum cystatin-c and urinary HO-1. Mutant genotype AA was associated with significant increase in urinary HO-1 levels, while heterozygote genotype GA was associated with significant fall in eGFR levels in diabetic subjects compared with control ones.Table 1C (%) and serum glucose in all ACE I/D polymorphism. Also, DD polymorphism was associated with significant increase in levels of serum creatinine and cyst-c in diabetic patient group. Moreover, I/D polymorphism was associated with significant increase in urinary HO-1 levels and high significant decline in eGFR levels in diabetic patients compared with control group.Table p < 0.027).Figure p < 0.02).Figure p < 0.001) to predict early tubular injury in diabetic kidney, suggesting that urinary HO-1 could represent as potent biomarker of DN in urine.Figure p < 0.005) for predicting early tubular injury in diabetic kidney suggesting that serum cystatin-c could be a potential biomarker of DN.Figure 21. Our study had focused on many markers such as serum cystatin-C and urinary hemeoxygenase to be screened as early indicators of DN in long-term type 2 diabetic patients.Diabetic kidney disease (DKD) accounted for the increased risk of mortality among diabetic patients and eventually was recorded in about half of diabetic patients with type 2 diabetes mellitus (T2DM)6. Also, other study confirmed that cystatin-c is a well-defined marker which is advised to be assessed for early DN diagnosis and progression22. As serum creatinine was unable to detect early decline in GFR owing to the fact that levels of serum creatinine begins to rise above normal range only in case of kidney had lost approximately 50% of its function. This proposes that serum cystatin-c levels are related to impairment in tubular function and in turn can be suggested as an early marker of kidney diseases23. Moreover, detecting CKD at early stage could be important for early intervention in order to be able to slow kidney function loss, which in turn helps improving survival and quality of life.Significant rise in serum cystatin-c levels with significant decline in eGFR in diabetic group was recorded in current study in comparison with control group. Our results were in accordance with Saptoka et al.14 who referred that hyperglycemia and diabetic substrates in the form of glycation end products can affect renal tubular cells. That results tubular cell hypertrophy and interstitial deposition of chemokines, cytokines and adhesion molecules leading to tubular inflammation and fibrosis24. HO-1 protein was upregulated in renal tubules but not in glomerular cells in affected (diseased) kidney. This upregulation may be due to the difference in both sensitivity and response to oxidant stress exhibited by these cells14.Regarding urinary HO-1, our results were in line with Abd ElHameed and Saied25 reported that patients with AA genotype were at lower risk for CKD in comparison with those with GG genotype. In current study, A-allele exhibited lower significant frequency in diabetic cases.Our results demonstrated significant difference in genotypic and allelic frequencies distributions of ACE I/D and G2350A polymorphisms between diabetic and control groups with dominant heterozygote polymorphisms were shown in both types of genes (I/D and GA) in diabetic patient group Table 26. While mutant AA genotype was associated with increase in urinary HO-1 that may confirm, as Narita et al.27 stated that higher risk for progressive renal dysfunction may be found in patients with AA genotype of the G2350A polymorphism. Furthermore, heterozygote genotype GA was associated with significant fall in eGFR levels in diabetic subjects compared with control ones.Present study recorded an association of GG genotype of ACE G2350A gene in diabetic patient group with rise in both serum cystatin-c and urinary heme oxygenase levels. Another study showed significant association between the G allele for the ACE G2350A gene polymorphism with higher blood pressure as well as ACE concentration. This association could induce nephropathy18, DD polymorphism was associated with DN and T2DM progression. This may be attributed to the functional role of DD polymorphism in the implication of pathological mechanisms through change of endothelial β cells, oxidative stress and renal microvascular complications. Also, Rahimi28 suggested that ethnicity represents one of the most important factors in determining ACE polymorphism role in DN susceptibility.Regarding ACE I/D polymorphism, the current study recorded that DD polymorphism was associated with increase in serum creatinine and cyst-c in diabetic patient group. According to Silveira et al.29 who revealed that in diabetic patients, ID polymorphism showed a slope in eGFR in T1DM and can be considered a determinant markers of DN progression. On the other hand, intermediate serum ACE activities were shown in heterozygous individuals (ID). That explained the association of higher ACE activity in patients with D allele with more extensive kidney damage30.Heterozygous ID polymorphisms was associated with significant fall in eGFR levels in diabetic individuals compared with controls which is in accordance with Sauca et al.p < 0.027). That was in accordance with Saptoka et al.6 who found the same relation.The current study found positive relation between duration of diabetes and level of serum cystatin-c in diabetic patient group (p < 0.02). That may be due to oxidative effect of hyperglycemia in diabetic patients. This could be explained by tubular injury resulted from hyperglycemia as mentioned in Chang et al.31 who referred proximal tubule injury to high glucose transport state and mismatched local hypoxia in DM patients might be initiating factors to proximal tubular injury.In addition, positive relation was found between urinary hemeoxygenase and serum glucose in diabetic patient group  of serum cystatin-c is 0.617, suggesting that serum cystatin-c could be blood biomarker that could predict early tubular injury in T2DM diabetic patients that is in line with Saptoka et al.We can suggest the need of larger population for confirming the cut-off value of either blood cystatin-c or urinary hemeoxygenase for predicting DN unlike serum creatinine.Findings of this study indicated an association of serum cystatin-c with GG genotype of ACE G2350A in conjugation with DD polymorphism of ACE I/D gene as early predictor of tubular injury in T2DM diabetic. In addition, an increase in cystatin-c level was seen with individuals with longer history of diabetes. Therefore, assessment of serum cystatin-c seems to be a better diagnostic test for screening patient when serum creatinine level is inconclusive, especially, in certain individuals with longer duration of diabetes and other comorbidity.This cross-sectional controlled study recruited 193 participants. They are grouped into 108 diabetic patients from out patient diabetes clinic in Kasr Al-Aini hospital and 85 controls. Control subjects were administrative and/or employee in different sectors in the National Research Centre in addition to other retired participants. The research protocol was approved by the Ethical Committee in the National Research Centre, Egypt with number (13132032021) in accordance with the Declaration of Helsinki. All the selected subjects signed an informed written consent for their participation in the study. A questionnaire including demographic, clinical and environmental history was filled out.1C) and ACE genes determination.Seven ml of fasting blood sample was collected from each participant and divided into 2 tubes, an EDTA tube was used for determination of glycated hemoglobin  was performed by ion exchange method according to Bates33 according to the protocol supplied from MG, Science &Technology Center.HbASerum cystatin-c levels was measured using an enzyme-linked immunosorbent assay (ELISA) commercial kit .Urinary hemeoxygenase levels was measured using an enzyme-linked immunosorbent assay (ELISA) commercial kit .34. Urine creatinine was estimated to adjust the values of the urinary hemeoxygenase.Determination of serum and urinary creatinine (Cr) were carried out by using the Jaffe kinetic method without deproteinization according to Bartels2) = 141 × min α × max −1.209 × 0.993Age × 1.018 × 1.159(if black) .Estimated GFR was calculated using Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equation; GFR , 3 mM MgCl2, 50 mM KCI, 10 mM Tris-HCI pH 8.4. Amplification program  was 30 cycles  while in the presence of the insertion (I), a fragment of 490 bp was observed. A heterozygote, third fragment, with an intermediate molecular weight showing both fragment at 190 bp and 490 bp corresponds to ID genotype. The patients were then grouped into the DD, II and ID genotypes respectively.ACE gene (I/D) polymorphism was performed by PCR methods according to Rigat et al.37. The PCR primers sequences were as follows:(ACE) gene G2350A was assayed by PCR-RFLP according to Mahmood et al.Forward primer: 5′-CTGACGAATGTGATGGCCGC-3′ and Reverse primer 5′-TTGATGAGTTCCACGTATTTCG-3′. The PCR conditions was carried out: initial denaturation at 95 °C for 5 min followed by 35 cycles where 30 s for denaturation at 94 °C, 30 s at 58 °C for annealing, and 30 s at 72 °C for extension, and 10 min at 72 °C a for final extension time. The PCR products were digested with 5 U of fast digest BstU1 at 60 °C for 2 h. Digested fragments were separated by electrophoresis on 3.5% agarose gel and identified by ethidium bromide staining using ultraviolet trans-illumination. Allele G2350 was visualized as a 122-bp fragment and allele A2350 as 100-bp and 22-bp fragments and GA genotype appear as 122–100 and 22 bp.DNA was extracted from peripheral blood mononuclear cells using using a QIAamp® DNA Blood Mini kit , according to the manufacturer's protocol for genetic assay:t-test. Significance was set at p < 0.05. Relation between variables was studied by Pearson's correlation. The χ2 test was used to examine the differences in genotype distribution between patients and controls groups. Receiver Operating Characteristics (ROC) analysis was done for calculating the Area Under the Curve (AUC) for serum cystatin-c and urinary HO-1 to find the best cut-off values to identify diabetic Nephropathy.Analysis of data was performed through SPSS program version 18. Expression of data was as mean ± SD. Differences between groups was compared by using Independent student’s The study was conducted in accordance with the Declaration of Helsinki, and ethical approval was obtained from National Research Centre Ethical committee (NRCEC-13132032021) of National Research Centre, Egypt.Informed consent was signed from all participants included in the study.


Hypertension is a major risk factor for heart attack, produce atherosclerosis (hardening of the arteries), congestive heart failure, stroke, kidney infection, blindness, end-stage renal infection, and cardiovascular diseases. Many mechanisms are involved in causing hypertension, i.e., via calcium channels, alpha and beta receptors, and the renin-angiotensin system (RAS). RAS has an important role in blood pressure control and is also involved in the metabolism of glucose, homeostasis, and balance of electrolytes in the body. The components of RAS that are involved in the regulation of blood pressure are angiotensinogen, Ang I (angiotensin I), Ang II (angiotensin II), ACE (angiotensin-converting enzyme), and ACE 2 (angiotensin-converting enzyme 2). These components provide for relevant therapeutic targets for the treatment of hypertension, and various drugs are commercially available that target individual components of RAS. Angiotensin receptor blockers (ARBs) and ACE inhibitors are the most popular among these drugs. ACE is chosen in this review as it makes an important target for blood pressure control because it converts Ang I into Ang II and also acts on the vasodilator, bradykinin, to degrade it into inactive peptides. This review highlights various aspects of blood pressure regulation in the body with a focus on ACE, drugs targeting the components involved in regulation, their associated side effects, and a need to shift to alternative therapy for putative hypertension treatment in the form of bioactive peptides from food. Hypertension accounts for 30–45% of the general population of which 60% are in the 60-65 years age group [Hypertension or high blood pressure, generally called arterial hypertension, is defined by the American Heart Association as persistent high blood pressure consistently higher or above 140/90 mm Hg with systolic 130 mm Hg or higher and diastolic 80 mm Hg or higher affecting billions of people and is a common factor of death . Hypertege group . To treage group . Primaryα1-adrenergic receptors [+ channels are listed , decreasing inflammatory markers, inhibiting muscle proliferation, extracellular matrix formation, and inhibition of apoptosis of endothelial cells –22. Amlovailable . The β-bhibitors , 27. Bethibitors .1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10); this product is then cleaved by circulating and locally articulated ACE to an octapeptide, Ang II  [RAS is a central target for renal and cardiovascular defense and electrolyte balance . Hormonao7-Phe8) , 34. Theo7-Phe8) . Antihypo7-Phe8) . ARBs aro7-Phe8) . The moso7-Phe8)  and haveo7-Phe8) .Evidence suggests that chronic administration of ARBs to mice causes a significant reduction in vascular tone by activating eNOS-derived NO, even at doses below those that lower blood pressure. This effect was not seen after similar ACE inhibition or in eNOS-null animals. Telmisartan, an acute ex vivo pleiotropic ARB with AT1R-independent signalling properties, also inhibits aortic contractility in eNOS-dependent ways that are unrelated to traditional Ang II/AT1R signalling .The angiotensin-converting enzyme commission number is 3.4.15.1. ACE expression is found in the capillaries of the lungs and also in the endothelium of the kidney. It is necessary for the regulation of blood pressure and electrolyte homeostasis throughout the renin-angiotensin system . RAS is ACE inhibitors are usually administered orally, although intravenous formulations are available. The most common suffix for these medications is “-pril.” Some of the examples are lisinopril, ramipril, and captopril. According to the chemical composition, it is divided into three groups: sulfhydryl-containing ACE inhibitor (captopril), phosphorus-containing ACE inhibitor (fosinopril), and dicarboxylic-containing ACE inhibitors  . ACE inhInhibition of the ACE leads to bradykinin accumulation in the lung . In humaOn the basis of decades of clinical observations on the use of ACEIs, a number of adverse effects have gained increasing attention. Interestingly, respiratory side effects have been a problem for a long time when ACEIs are used. A dry cough is a major side effect of respiratory side effects , 55. SomTwo predominant forms of ACE are articulated in humans; these are somatic form and germinal form. The somatic form is abundantly found on the endothelial surface of lung vessels and other cell types and also on the surface of monocytes, smooth muscle cells, T lymphocytes, and adipocytes, whereas the germinal form is present exclusively in the testis. Both forms of ACE are present on the outer cell surface as ectoenzymes, where they hydrolyze circulating peptides. The soluble form of ACE, different from the membrane-bound form, is formed due to the ACE secretase action and is found in serum and other body fluids. Testicular ACE is the ancestral form of the molecule with a particular active site; its crystal structure was published in 2003 . SomaticSomatic ACE (sACE) has a significant role in the blood pressure regulatory mechanism (renin-angiotensin system). In somatic ACE, the polypeptide chain is 1,277 amino acids in length . It is iTesticular ACE has an important role in reproduction and is 701 amino acids in length . It has ACE 2 is a zinc metalloenzyme and carboxypeptidase situated as an ectoenzyme on the surface of endothelial and disparate cells. It has a great homology with ACE and catalyzes the cleavage of angiotensin I directed toward angiotensin 1-9, and angiotensin II into the vasodilator angiotensin 1-7 as demonstrated schematically . AlthougNumerous studies regarding polymorphisms in genes associated with RAS have been established. These polymorphisms have been found to be associated with various cardiovascular diseases . PolymorAngiotensin converting enzyme is the main component of the renin-angiotensin system, and its insertion and deletion polymorphism is reported in intron 16 , 82. ManCommercially available antihypertensive drugs are given as a single drug or in combination of more than one drug so as to regulate high blood pressure. Although these drugs are very potent in controlling blood pressure in hypertensive patients, they have a number of side effects, some common and others rare. Some of these effects include low blood pressure or hypotension, kidney failure, liver failure, and decreased white blood cell count. These antihypertensives may also lead to birth defects in pregnant women and therefore should not be consumed during pregnancy. Studies have been conducted on the effects of antihypertensive drugs apart from their blood pressure-lowering effects.A study carried out by Schunkert et al. showed that patients consuming antihypertensive like diuretics, beta blockers, ACE inhibitors, and ARBs are at a high risk of gout. Beta blockers and diuretics also increase the serum levels of uric acid which is in turn associated with gout . AnotherAllium sativum (garlic), Andrographis paniculata (king of bitter), Apium graveolens (celery), Bidens pilosa L. , Camellia sinensis (tea), Coptis chinensis (goldthread), Coriandrum sativum (cilantro or coriander), Crataegus spp. (hawthorns), Crocus sativus (saffron), Cymbopogon citratus (lemongrass), Hibiscus sabdariffa (roselle), Nigella sativa , Panax (ginseng), Salviae miltiorrhizae (chinese sage), and Zingiber officinale (ginger) [Stevia leaf for treating diabetes and hypertension [Drugs extensively used for the treatment of hypertension cause a plethora of side effects, some rare and others serious, as mentioned in the above section. The need therefore arises to consider other sources of therapy for hypertension in the form of herbal medicines that have few or no known side effects  and are (ginger) . A studyrtension . These artension .Apart from the antihypertensive plants mentioned above, honey has also been shown to have therapeutic effects on cardiovascular diseases. People have been using honey since time immemorial for treating a plethora of ailments, and researchers have just started reconsidering its role in nutrition and therapeutics . The higBioactive peptides that are derived from food are compounds originating from animals and plants that are produced by food processing and fermentation . Apart fThe length of inhibitory peptides is usually short, about 12 amino acids in length. This short length has its advantages for the inhibitory activity of the peptide such as helping in absorption and thereby ensuring its bioavailability. Some studies have reported that the presence of certain conditions enhance the ACE binding ability of the peptides. These include the presence of certain amino acids (hydrophobic) at the C-terminal along with the presence of proline, leucine, tryptophan, phenylalanine, and tyrosine at the C-terminal of the peptide. ACE inhibitory bioactive peptides have three classifications, namely, inhibitor type, substrate type, and prodrug type on the basis of their mode of action . In the An interesting study on human subjects showed that oral administration of milk-derived peptides, IPP and VPP, significantly reduced blood pressure levels in individuals of Finnish and Japanese racial origin, but the same peptides failed to do so in individuals of Danish and Dutch racial origin. When these studies are performed in animal models, the animals usually belong to a specific strain, whereas human subjects may be of different racial and genetic origin. This leads to discrepancies in the results and efficacy of bioactive peptides. Therefore, this creates a need for more and more clinical trials on human subjects of varied ethnicities to establish a solid ground for the antihypertensive nature of bioactive peptides .The present review highlights the role of the angiotensin-converting enzyme in blood pressure regulation and how its inhibitors are used in the control of hypertension. From our review, we can safely conclude that commercially available synthetic ACE inhibitors pose numerous side effects in patients, and since hypertension is a chronic disease and leads to various cardiovascular disorders, the long-term consumption of these synthetic inhibitors can be harmful.Therefore, based on evidences from existing literature, it can be concluded that natural remedies in the form of medicinal plants, bioactive peptides from food, and honey can be used as putative treatment option for hypertension and other cardiovascular diseases. Further research is definitely required in this direction to accept or reject these remedies for their antihypertensive nature.


Anorectal manometry measurements exhibit significant interrater variability. Newer techniques like 3D high-resolution anorectal manometry (3D-HRAM) have the potential to enhance diagnostic accuracy and our understanding of defecation disorders. However, the extent of interrater variability in 3D-HRAM is still unknown. Between January 2020 to April 2022, patients referred for pelvic floor physical therapy (PFPT) due to functional defecation complaints underwent 3D-HRAM testing. In a retrospective analysis, three expert raters independently evaluated the 3D-HRAM results in a blinded matter to assess interrater agreement. The evaluation also determined the level of agreement concerning dyssynergic patterns during simulated defecation. The 3D-HRAM results of 50 patients  were included. Twenty-nine patients had complaints of fecal incontinence, eleven patients had chronic constipation, and ten patients had several other complaints. There was a substantial agreement (kappa 0.612) between the raters concerning the 3D images on dyssynergic patterns during simulated defecation. Our study emphasizes the need for standardized guidelines in evaluating 3D-HRAM test results to reduce subjectivity and further improve agreement among raters. Implementing these guidelines could improve diagnostic consistency and enhance personalized treatment strategies, increasing the reliability and usefulness of 3D-HRAM testing in clinical practice. Chronic constipation and fecal incontinence are functional defecation disorders that affect people of all ages and are associated with a reduced quality of life ,2. TogetFunctional defecation disorders present several overlapping symptoms, thereby complicating accurate diagnosis and potentially resulting in a wide spectrum of treatment modalities accompanied by significant challenges. It can be time-consuming and frustrating for both the patient and the treating specialist; therefore, it is essential to correctly identify the underlying cause.Conservative treatment that includes lifestyle changes, exercise, an increase in the intake of dietary fiber, laxatives, and pelvic floor physical therapy is usually sufficient to reduce or eliminate symptoms in the majority of patients . HoweverFunctional defecation disorders are defined by the Rome IV criteria as disordered evacuation resulting from an inadequate recto-anal pressure gradient brought on by paradoxical contraction or insufficient relaxation of the pelvic floor muscles and/or insufficient rectal propulsive forces during defecation ,9. DyssyDiagnostic tests such as three-dimensional high-definition anorectal manometry (3D-HRAM) or defecography can provide valuable insight into the underlying cause of anorectal dysfunction and guide the development of a tailored treatment plan. Which anorectal function test is the most accurate is still under debate. In our previous study, we demonstrated that for detecting dyssynergic defecation, there is no correlation between 3D-HRAM interpretation and other anorectal function tests such as DRE (52% agreement), transperineal ultrasound (25% agreement), s-EMG (52% agreement), or a balloon expulsion test (46% agreement) . Other sARM is a diagnostic tool used to evaluate the function of the rectum and anal sphincter muscles in an objective manner. It involves inserting a catheter into the rectum and measuring the pressure and sensation in the anal canal at rest, during squeezing, and simulated evacuation. Traditional anorectal manometry uses a single sensor to measure pressure, while 3D-HRAM uses an array of 256 sensors, providing a more detailed evaluation of the anorectal anatomy and function thanks to its 3D pressure profile map. 3D-HRAM is a valuable diagnostic tool for evaluating pelvic floor dysfunction in patients experiencing obstructive defecation or fecal incontinence . It can Despite its usefulness in evaluating anorectal function, measurements obtained during ARM can be subject to substantial external influence ,17. A re3D-HRAM has been shown to enhance diagnostic gain by visualizing puborectal muscle function and identifying focal sphincter defects . DespiteThe question of whether we can rely on 3D-HRAM results to interpret anorectal function and guide treatment strategies is complex, as both operator variability and interrater variability can affect the reliability and diagnostic accuracy of the test ,22,23. TThis retrospective study was conducted at a Dutch tertiary referral clinic specialized in proctology, where a multidisciplinary team consisting of surgeons, gastroenterologists, pelvic floor physiotherapists, and specialized nurses work closely together in the treatment of patients with defecation disorders. Recently, we published the findings of a study by Dekker et al., which involved fifty patients who consented to participate . These pPatient data including demographics, complaints, previous perianal surgical procedures, obstetric history, and history of previous pelvic floor physical therapy were collected. To evaluate the interrater agreement of 3D-HRAM results, three experienced raters (two surgeons and one gastroenterologist) independently evaluated the results of fifty patients. To ensure unbiased evaluation, the raters were blinded to the initial reports and to the evaluations of the other raters. To evaluate intra-rater agreement, two raters evaluated several cases at two different time points: once as part of the diagnostic process and once for the present study, with a minimum interval of 6 months between these two evaluations. The raters were also blinded to their original reports to prevent any bias in the evaluation.For 3D-HRAM, the equipment consists of the following components: a probe, a pressure recording device, a monitor, and a computer used for data storage. The 3D-HRAM probe is equipped with 256 pressure sensors on 16 lines, with each line containing 16 circumferential sensors. It is covered by a disposable sheath, measuring 10.75 mm in diameter and 64 mm in length, which has an inflatable balloon (capacity 400 cc) at the most distal end.Two experienced continence nurse specialists operated the 3D-HRAM in a protocolized matter, following the International Anorectal Physiology Working Group (IAPWG) protocol . The insThe software calculated the mean resting pressure (MRP) and maximal squeeze pressure (MSP) in each patient, which are standard parameters in HRAM manometry. Meanwhile, the 3D-HRAM-derived images were collected and compiled into a single database with all patient identifiers removed. Visual representations of the changes in anal and rectal pressure during the protocol maneuvers are presented through color patterns and 2D and 3D images . The preThe color contour plots of the 2D- and 3D-HRAM images were evaluated by three independent raters  and categorized as low, normal, or high pressure, as indicated in During simulated defecation (straining), we measured several parameters including mean anal resting pressure, percentage of anal relaxation, intra-rectal pressure, and recto-anal pressure gradient (RAPG). Additionally, we calculated the percentage of positive RAPG, which could suggest adequate rectal propulsive forces during simulated defecation. Furthermore, the three raters independently evaluated the 2D and 3D images for the presence of a dyssynergic pattern of defecation during straining, defined as paradoxical anal contraction , inadequate relaxation of the resting anal sphincter pressure, or inadequate effort of the abdominorectal muscles . In normFinally, the presence of slow waves and ultraslow waves (USWs) was evaluated visually by two of the three independent raters. These two raters conducted a visual evaluation and classification of maximum tolerated volume (MTV) sizes, along with the measured values of the first constant sensation volume (FCSV) and desire to defecate volume (DDV). This analysis aimed to determine whether these factors contributed to any sensory disorders.p < 0.05. Statistical analyses were performed using SPSS software . Continuous data are reported as either median or mean, depending on the distribution, accompanied by the range or standard deviation. For categorical outcomes from the 3D-HRAM evaluation, descriptive statistics and crosstabs were used for comparison. The interrater agreement among the different raters for various 3D parameters, including mean resting pressure, maximum squeeze pressure, dyssynergia, presence of (ultra)slow waves, rectal volume, and sensibility, was evaluated using Cohen’s Weighted Kappa test . The agrA total of 50 patients  with a median age of 51 years were included in this analysis. The indication for 3D-HRAM differed for these 50 patients: 29 patients had complaints of fecal incontinence, 11 patients had chronic constipation, and 10 patients had various other anorectal complaints . Thirty The descriptive statistics of the 3D-HRAM software-derived data of the pressure variables in the study group are detailed in p < 0.001). However, for maximum squeeze pressures, the agreement was only deemed fair, with a mean kappa of 0.329 (p < 0.001). The evaluation of a dyssynergic pattern during three straining maneuvers (simulated defecation) resulted in a substantial level of agreement, with a mean kappa of 0.612 (p < 0.001). In terms of (ultra)slow waves and MTV size and sensibility, the agreement was found to be poor, almost perfect, and substantial, respectively , we observed moderate or fair agreement, respectively. These findings indicate that there is a significant amount of variation in the interpretation of 3D-HRAM results.The variability in agreement regarding conventional HRAM results might be attributed to the reliance on software-derived values, which often leads to the interpretation of mean pressures at rest and during squeezing and straining. However, when examining the 3D images, it becomes evident that there are regions of high pressure in a specific area of the anal sphincter, indicating hypertonia, while simultaneously showing a defect in another area, resulting in low pressure at that specific point. This information is crucial because solely looking at mean pressure values can thus lead to the incorrect conclusion that there is no pathology, potentially affecting the treatment strategies and conclusions drawn for patients.The evaluation of a dyssynergic pattern during three subsequent straining maneuvers demonstrated substantial agreement among the three raters. However, challenges were observed in visually evaluating dyssynergia in patients with low anal resting tones, probably due to less pronounced patterns. Unlike previous studies, the raters categorized the presence of a dyssynergic pattern as either yes or no, without using different categories or phenotypes ,20. PrevIn the literature, ultraslow waves are defined as repeated pressure oscillations occurring at a rate of 1–2 per minute with an amplitude >25 mmHg, while slow waves occur at a rate of 10–20 per minute and are also widely present in healthy people ,14. The n = 11), we observed almost perfect agreement among raters regarding dyssynergic defecation patterns (kappa = 0.855). Therefore, it is crucial to create a clear overview of different patient groups and their symptoms, incorporating 3D-HRAM results to gain a better understanding of the underlying pathophysiology. It is important to note that in this specific test cohort, the raters were intentionally blinded to patients’ history, complaints, and digital rectal examination findings. The aim was to solely evaluate interrater agreement based on the 3D-HRAM results. However, in real-world diagnostic scenarios, this additional information would be considered and integrated with the 3D-HRAM results, providing a comprehensive and more accurate diagnostic evaluation.Our study suggests that a more homogeneous patient population leads to higher agreement levels among raters. In a sub-analysis of 3D-HRAM results from patients suffering from chronic constipation , with each rater performing evaluations in their own individual manner without a standardized approach. Additionally, intra-rater data was available for only a subset of patients (n = 39). Since there was no established gold standard, reference test, or consensus agreement, it was challenging to compare the outcomes and accuracy of the three evaluations. This study is the first to evaluate the agreement among experienced raters in a blinded and independent manner regarding 3D-HRAM test results for patients with functional defecation disorders. However, it is important to acknowledge the study’s limitations. We report on a retrospective sub-analysis of a single-center modestly sized cohort (The agreement regarding dyssynergic patterns in 3D-HRAM is significant. Looking ahead, there are promising prospects for utilizing 3D-HRAM in other medical indications. Notably, it has shown potential in the field of fistula diagnostics . In the This study revealed that the current approach for the evaluation of 3D-HRAM test results, including the interpretation of 3D images, is subjective and leads to significant disagreement among experienced raters. Therefore, it is crucial to establish standardized guidelines for evaluating 3D-HRAM test results, preferably incorporating patterns categorized by specific functional phenotypes. This approach can help clinicians minimize subjectivity and enhance the uniformity of diagnoses. This standardization has the potential to enhance treatment strategies, ensuring they are more personalized to meet the individual needs of patients. Ultimately, implementing standardized guidelines may increase the reliability and usefulness of 3D-HRAM testing in clinical practice.


No significant effect of each tumor therapy-related factor on masticatory function was detected. A logistic regression analysis identified the number of functioning occlusal supports as a significant predictive factor. These results implied the crucial interactions of masticatory function with various factors and the specificities of maxillectomy patients.The aim of this retrospective cross-sectional study was to comprehensively assess masticatory function in maxillectomy patients with functioning removable prostheses. Their general and oral profiles, the measurement values of their oral functions, including masticatory function, and the history of tumor therapy were extracted from medical charts. The correlations of masticatory function with numerical data and the effects of tumor therapy-related factors on masticatory function were evaluated. In addition, a stepwise conditional logistic regression analysis was performed to identify the potential predictive factors comprehensively. The data from 55 maxillectomy patients revealed that the median value of masticatory function (138.0 mg/dL) was higher than the threshold (100.0 mg/dL) based on the concept of oral hypofunction. Moderate correlations of masticatory function with the number of remaining teeth, the number of functioning occlusal supports, and maximum occlusal force were found, as well as a weak correlation with maximum tongue pressure. These variables also showed statistically significant coefficients ( Maxillary resection, or “maxillectomy,” is conducted to ablate benign or malignant tumors as a radical treatment. Maxillectomy results in structural and functional alterations, and psychological distress. In particular, oral functions including mastication, deglutition, and speech are often impaired by maxillectomy, and reconstructive and/or prosthetic rehabilitation can be therapeutic options to restore oral functions ,2,3,4,5.Multiple oral functions and patient profiles have been reported to be associated with masticatory function in the healthy elderly population ,12,13,14The aim of the present study was to comprehensively assess masticatory function in maxillectomy patients with functioning removable prostheses in surgically resected areas. In other words, tumor therapy-related factors such as ablative surgery, radiotherapy, and chemotherapy, patient profiles such as the number of remaining teeth and functioning occlusal supports, and the measurement values of their oral functions were adopted as potential predictive factors. Oral functions were evaluated using the methods for the diagnosis of “oral hypofunction,” which are well known in Japan ,26. Our This retrospective cross-sectional study was conducted at the Department of Prosthodontics, Kyushu University Hospital, Fukuoka, Japan. This study was approved by our institutional review board for clinical research . This study was conducted in compliance with the Helsinki Declaration and the Strengthening the Reporting of Observational Studies in Epidemiology guidelines.The candidates for this study were maxillectomy patients with or without reconstructive surgeries who had visited our department from April 2014 to October 2022.Patients with maxillectomy due to benign or malignant tumors;Patients after prosthetic rehabilitations in surgically resected areas using removable prostheses without any implants and without any problems in their removable prostheses at the time of evaluation of their oral functions;Patients with the data of their oral functions that had been evaluated when they had used their removable prostheses in surgically resected areas without any problems.Inclusion criteria were as follows: Patients with maxillectomy due to other diseases (except for tumors);Patients without prosthetic rehabilitations, with prosthetic rehabilitations in surgically resected areas using fixed prostheses or implant-assisted prostheses, or with difficulties in the usage of their removable prostheses in surgically resected areas;Patients without data on their oral functions.Exclusion criteria were as follows:The following data were extracted from a medical chart.The data for subjects’ profiles include age, gender, primary tumor, and the number of remaining teeth and functioning occlusal supports. All data at the time of oral function evaluations were collected. The number of functioning occlusal supports was defined as the number of antagonistic occlusal contacts by natural teeth and fixed prostheses, including pontics. However, the occlusal contacts made by artificial teeth in removable prostheses were not counted. Oral functions were evaluated based on the concept of oral hypofunction ,26. ThesMasticatory function: masticatory function was defined by the glucose concentration in 10 mL water from the particles of crushed gummy jelly (2 g) after 20 s voluntary chewing. Commercially available devices (Glucoram (a gummy jelly) and Gluco Sensor GS-II, GC Co., Tokyo, Japan) were adopted as described previously ,15, and Oral functions and the measurement methods were as follows:2.Oral moisture (OM): OM was measured using an oral moisture checker  . Briefly3.Maximum occlusal force (MOF): A pressure-sensitive sheet  was used to record MOF after maximum clenching and was analyzed with a dedicated software  . The thr4.Tongue–lip motor function; oral diadochokinesis (ODK): tongue–lip motor function was defined by ODK . The rep5.Maximum tongue pressure (MTP): MTP was measured using a commercially available device  . BrieflyThe history of tumor therapies includes the extent of the resected defect , the existence or non-existence of soft palate resection, oronasal and oroantral communication, radiotherapy, chemotherapy, and neck dissection.p < 0.05 was considered statistically significant.Numerical data from medical charts are described as median values and interquartile ranges (IQRs). Spearman correlation coefficients (ρ) were calculated between masticatory function and other numerical data  to validate the possible correlations. The Wilcoxon rank sum test was used to compare masticatory function between the subjects with and without the histories of their tumor therapies. A stepwise conditional logistic regression analysis was performed to verify the potential predictive factors using multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (CIs). Numerical data were classified into 2 groups according to the median values  or the threshold value of each factor for a stepwise conditional logistic regression analysis and calculation of odds ratios. The JMP16 software  was used for all statistical analyses, and a value of The present study included 55 subjects . They had received maxillectomy due to adenocarcinoma (2 subjects), adenoid cystic carcinoma (4 subjects), mucoepidermoid carcinoma (2 subjects), squamous cell carcinoma (38 subjects), chondrosarcoma (1 subject), oral melanoma (1 subject), pleomorphic adenoma (1 subject), ameloblastoma (1 subject), and 5 subjects had been unknown (no record). Maxillectomies were classified into 3 categories: limited maxillectomy or partial resection (47 subjects), subtotal maxillectomy (4 subjects), and total maxillectomy (4 subjects), and extents of resected defects were classified into two categories, unilateral (31 subjects) and bilateral (24 subjects), which were adopted as tumor therapy-related factors. The median value and IQR of each numerical variable are shown in p < 0.01).The correlations between masticatory function and other numerical data are presented in The effects of tumor therapy-related factors on masticatory function were evaluated by comparing two groups . Although the median values of masticatory function in the subjects with soft palate resection, oronasal and oroantral communication, chemotherapy, and neck dissection, meaning more intervention groups, were higher compared to the subjects without these interventions, unexpectedly, these analyses revealed that no tumor-related therapies induced significant differences in masticatory function .Including all factors analyzed in this study , a stepwise conditional logistic regression analysis was conducted. As described above, all subjects were classified into two groups according to median values: the threshold value of each oral function and the history (with or without) of each tumor therapy. The factors selected in a stepwise method were the number of functioning occlusal supports and OM, and multivariable-adjusted ORs are shown in Mastication is the process of forming a bolus for deglutition and digestion using teeth, saliva, tongue, masticatory muscles , and the neuromuscular system (mandibular motor function) . PreviouTumor therapies, including surgical resection, radiotherapy, and chemotherapy, cause multiple complications. In particular, maxillectomy for oral or head and neck tumors results in the loss of anatomical structures and impaired physiologic functions. Masticatory function is one of the most affected functions by maxillectomy, and surgical reconstruction and/or prosthetic rehabilitation are necessary to restore this function. However, maxillectomy patients who had received multiple tumor therapies might possess various conditions , and thiAt first, oral functions were evaluated based on the concept of oral hypofunction ,26. The Second, the correlations between masticatory function and numerical data, including the numbers of remaining teeth and functioning occlusal supports, and oral functions were analyzed using Spearman rank correlations. These analyses showed that the numbers of remaining teeth, functioning occlusal supports, and MOF exhibited moderate correlations with masticatory function. In addition, MTP exhibited a weak correlation. All correlations were statistically significant. These results were consistent with previous studies ,19,20,29Third, the effects of tumor therapy-related factors on masticatory function were evaluated. The present study indicated that no significant difference between masticatory function in the subjects with and without each therapy was identified, although some groups with more interventions showed higher median values of masticatory function. According to the previous studies, some tumor therapy-related factors, such as the extent of the resected defect ,17,19,29Lastly, based on the presence of multiple variables and potential interactions, a stepwise conditional logistic regression analysis was performed to assess the predictive factors, including all numerical variables and tumor therapy-related factors, which were nominal variables. This analysis identified the number of functioning occlusal supports as a significant predictive factor. It would be reasonable if a greater number of functioning occlusal supports acted positively on masticatory function. As described above, some tumor therapy-related factors could have been predictive factors in previous studies ,17,19,29The present study comprehensively evaluated masticatory function and its influential factors in maxillectomy patients. Each individual factor, such as the number of remaining teeth and functioning occlusal supports, MOF, and MTP, showed a significant correlation with masticatory function. No significant effect of each tumor therapy-related factor on masticatory function was detected. A stepwise conditional logistic regression analysis suggested that the number of functioning occlusal supports plays a crucial role among potential variables. These results implied the specificities of maxillectomy patients.


Although effective antihypertensive medications have existed for decades, only about half of the hypertensive individuals are considered to have controlled blood pressure. Limited research studies have investigated gender disparity in the utilization and effectiveness of antihypertensive medications treatment. To examine the gender difference in antihypertensive medications’ use and the effect of using antihypertensive medication treatment on blood pressure control among the U.S. adult with hypertension. Analysis of National Health and Nutrition Examination Survey (NHANES) data from (1999-2012) including individuals≥18 years old with hypertension. Study variables included gender, age, race/ethnicity, obesity, smoking, comorbidities, treatment medication type, and continuity of care. We used multivariate logistic regression in STATA V14. The data is presented as adjusted odds ratios (ORs) and 95% confidence interval (CI). P<0.05). No association between gender and blood pressure control was found. Blood pressure control was less likely achieved among 50 years or younger individuals, Blacks and Hispanics, obese, and those taking calcium channel blocker (CCB). Of the 15719 participants, 52% were female. 49% of the antihypertensive medication users had their blood pressure under control (95% CI). In the adjusted logistic regression analysis, use of antihypertensive medications was found to be 12% greater in females as compared to males (OR=1.12; CI=1.02-1.22; Hypertensive females are more likely than males to use antihypertensive medications. The effectiveness of treatment to control blood pressure is equal across males and females. Our findings have implications for practitioners to account gender-specific approaches when discussing adherence to hypertension medication treatment with their patients. In the U.S., the annual healthcare spending related to hypertension was found to be $131 billion when averaged over 12 years, from 2003 to 2012.6 It is estimated that individuals with hypertension have annual healthcare costs of $2000, which is greater than their counterparts.6 Effective medication treatment to lower blood pressure, such as angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARBs), diuretics, beta-blockers, and calcium channel blockers (CCBs) have existed for several decades. However, only about 50% of Americans living with hypertension during 2011-2016 had their blood pressure under control.5 Moreover, there remains an issue with adherence to antihypertensive medications despite their efficacy in controlling hypertension and reducing cardiovascular events.8 Non-adherence is associated with many factors, including inadequate insurance,9 age, race/ethnicity, and income.8 Past studies have focused on the relationship between gender and the utilization and effectiveness of antihypertensive medications among the U.S. adults, yet gender-specific guidelines for the treatment of hypertension and blood pressure control goals remain unaddressed.11 The Systolic Blood Pressure Intervention Trial (SPRINT) elucidated the need to establish the more intensive treatment of hypertension cut-off goals for both genders, like a systolic blood pressure of ≤ 130 mm Hg. However, the optimal blood pressure for both genders was not obtained due to a lack of significant data that stemmed from low female enrollment rates .12 Their finding emphasizes the need to clarify the relationship between gender and hypertension to optimize the potential of reducing high blood pressure and subsequent cardiovascular events. This study aimed to examine the gender difference in antihypertensive medications’ use and the effect of using antihypertensive medication treatment on blood pressure control among the U.S. adult with hypertension. Hypertension is a well-recognized and modifiable risk factor for cardiovascular diseases (CVD), such as myocardial infarction, stroke, and heart failure, and subsequently CVD mortality.13 The NHANES data is a cross-sectional survey based on a nationally representative sample of the U.S. population and is sponsored by the Center for Disease Control and Prevention. The survey combines an in-person home interview followed by a physical examination, including biomarkers. Non-institutionalized US civilians, aged 18 and older, with hypertension, were eligible to participate in the survey. Following the 8th Joint National (JNC 8) Committee’s recommendation, hypertension was defined as a systolic value ≥ 140 mm Hg or a diastolic value ≥ 90 mm Hg.14 A participant was classified having hypertension if he/she met one of the following criteria: 1) had a mean systolic reading of ≥ 140 mm Hg or mean diastolic reading of ≥ 90 mm Hg measured during the physical examination. 2) The participant had been on antihypertensive medication within the last 30 days. 3) The participant recalled being previously diagnosed with hypertension by a physician. This cross-sectional study used the National Health and Nutrition Examination Survey (NHANES) data from 1999 to 2012. The physical examination for NHANES data took place at a local mobile examination center and included measurements of the participants’ blood pressure, height and weight, and blood and urine tests. Trained physicians following standard protocol obtained blood pressure measurements after the participants rested for five minutes in a seated, upright position. Mid-arm circumference was used to determine the appropriate cuff size for each participant. Three consecutive blood pressure readings were obtained, 30 seconds apart, and an average was calculated to determine the mean blood pressure for each participant. The outcome variables for the study were taking medications for blood pressure and subsequent controlled blood pressure. Blood pressure medication use and type were obtained from the participants during the home interview when they were asked “have you taken or used any prescription medicines for blood pressure in the past month?” If the participant answered yes to this question, the name, duration, and primary reason for each medication was obtained. Antihypertensive medications reported by the participants were classified into one of five categories: diuretics, ACE inhibitors, ARBs, beta-blockers, CCBs, and other antihypertensive medications . Blood pressure control was defined as a blood pressure reading of less than 140/90 mm Hg and was measured during the physical examination at the local mobile examination center. The primary predictor variable was gender, categorized as male or female. Other independent variables in the study include age ; race/ethnicity ; comorbidities including diabetes, chronic kidney disease, congestive heart failure (CHF), stroke, and coronary heart disease, obesity based on body mass index (BMI) (obese vs. non-obese); smoking status ; continuity of care, and antihypertensive medication type.15 statistical software to analyze the data. A descriptive statistical analysis was performed to determine the general characteristics of the study population. Categorical variables, such as age groups, were described with numbers and percentages, while continuous variables, such as blood pressure, were described with means and standard deviations. A bivariate statistical analysis was subsequently performed to display the study demographics by gender  using chi-square tests. A two-step multivariate statistical analysis was performed using adjusted logistic regressions. First, we identified predictors of antihypertensive medication use in the hypertensive participants. Then, we analyzed the status of blood pressure control among medication users. In both analyses, we controlled for the demographics and other characteristics of the study sample. We used adjusted odds ratios (ORs) and 95% confidence interval (CI) to report our findings and STATA V14®P < 0.01). Moreover, higher proportion of females in compared to males used CCBs (17% vs. 14%), diuretics (19% vs. 15%), and ARBs (9% vs. 8%) (P < 0.01). Females had a higher mean systolic blood pressure, while males had a higher mean diastolic blood pressure (P < 0.01).P < 0.01). Other independent predictors of using hypertensive medications were being 40 and older in comparison to the younger age groups (P < 0.01); being former smoker  or non-smoker  in compared to the current smokers, and being obese compared to non-obese . No statistical significance was found between race and being hypertensive in the sample population.P < 0.01), and kidney disease (P < 0.04), had higher odds of being on hypertensive medications compared to their counterparts without any comorbidity. These findings show a strong relation between hypertension and co-morbidities at confidence interval of 95%. Also, participants with comorbidity, including diabetes, CHF, CHD, stroke (P < 0.05). Furthermore, compared to the Whites, Blacks and Hispanics had lower odds of having their blood pressure controlled  (P < 0.01). Obese individuals were 1.23 times at greater odds of having controlled blood pressure than non-obese participants (P < 0.01). Also, individuals taking CCB were 14% less likely to have their blood pressure controlled than individuals not taking CCB (P < 0.05).16 These results may be explained by consistent gender differences noted in the literature regarding the utilization of health services and the use of medications. A continuous trend in the literature shows that women use health services more than men. Specifically, one study noted that women had a significantly higher number of mean visits to their primary care clinic and diagnostic services ordered along with higher associated medical service charges.17 Also, studies have found that women use more medications, across various medication types and disease states, than men.19 Thus, the higher odds of women using antihypertensive medications extend the current literature with an emphasis on hypertension. This finding may suggest the need for gender-specific approaches when discussing hypertension with patients to mitigate the gender difference seen in medication use. Thus, further studies should focus on the factors associated with or causing the reported gender disparity. Our study found that females were more likely to use antihypertensive medications relative to males. Moreover, we did not find any significant differences in blood pressure control between males and females. Our finding of gender differences parallels a previous study that analyzed the NHANES data set between 1999 to 2004 found women used antihypertensive mediations at a higher prevalence 61.4%, relative to men 56.8%. Additionally, we found that former smokers and lifetime non-smokers were more likely to be on antihypertensive medications than current smokers. Current smokers may avoid seeking medical attention to avoid talking to physicians regarding quitting smoking. Moreover, hypertensive individuals who were obese or had comorbidities had a higher likelihood of being on medications for hypertension. These results may allude to the possibility that these individuals were sicker with other conditions and were already seeing a physician for those conditions and were already taking antihypertensive mediations medications related to their respective comorbidities. For example, individuals with CHF and Stroke were probably on antihypertensive medications due to having a history or CHF and stroke.20 A study that used NHANES data from 1999 to 2004 and adjusted logistic regression, similar to the current study, found that blood pressure control rates were not significantly different between genders.21 Conversely, a study that also used NHANES data from 1999 to 2004 and adjusted logistic regression found hypertensive women to be less likely to achieve blood pressure control- 44.8% of women were noted to reach blood pressure control compared to 51.1% of men.16 One contributing factor to the discrepancy between these two studies may be the larger sample size of the latter study (N = 5410) compared to the former (N = 3475), which may have unveiled the difference between genders within the same timeframe. Other prior studies have varying results compared to our study as they have noted gender differences in blood pressure control- either women having higher rates of control23 or lower rates of control compared to men.25 Our findings that there are no gender differences in blood pressure control varies by study design, time-period, sample size, and data analysis, which provides a potential explanation to the conflicting results. These overall inconsistent findings throughout the literature emphasize the need for future studies to evaluate whether gender difference exists in blood pressure control.26 By doing so, healthcare practitioners can establish gender directed treatment plans for their hypertensive patients. Lack of any significant differences in blood pressure control between males and females parallels with some of the current literature. One study noted that almost all large, prospective, and randomized trials in the hypertension arena had found no significant gender differences in blood pressure reductions.28 and calls for the identification of multifactorial causes.29 Furthermore, there was no statistically significant difference between the use of different antihypertensive medications and blood pressure control, expect for CCB. The antihypertensive medication users who were taking CCB were less likely to achieve the targeted blood pressure than individuals not taking CCB. Additionally, Blacks and Hispanics were less likely, 68% and 82% respectively, to have controlled blood pressure when taking medications compared to Caucasians. This is consistent with current research findings Although this study relies on national data and has a large sample size, some limitations should be noted. While the NHANES methods to obtain blood pressure measurements followed national standard protocols and obtained three readings, each participants’ blood pressure was obtained in one sitting time rather than two or more occasions. This may have resulted in inaccurate participant blood pressure classifications. Moreover, to minimize recall bias, NHANES asked participants to recall antihypertensive medications taken in the past month. Therefore, participants who took antihypertensive medications prior to that one month window were classified as non-medication users and potentially non-hypertensive if they did not meet the other two criteria. Thus, the prevalence of hypertension among U.S. adults may have been underreported Our study concluded that, compared to males, females were more likely to adhere to the antihypertensive medication regimen. However, there were no gender differences in reaching the targeted blood pressure  among hypertensive individuals who were taking antihypertensive medication. Additionally, individuals older than 40 years, smokers, obese, and individuals with co-morbidity were more likely to use antihypertensive medication. However, blood pressure control was less likely to be achieved among hypertensive individuals who were 50 years or younger, Blacks and Hispanics, obese, and those taking CCB. Our findings suggest that providers may consider gender-specific approaches when discussing adherence to antihypertensive medication with their patients to mitigate gender disparity. Further studies are needed to verify our findings and perhaps shed light on why race-ethnic disparities in controlling hypertension continue to exist in the US. The research team would like to acknowledge the administrative and editorial support provided by Kaveh Dehghan, MBA.Health Promotion Perspective. Other authors declare no conflicts of interest in this work. Shahrzad Bazargan-Hejazi is an associate editor of  Data for this study is publically available for the readers. Not Applicable. Research for this article was supported in part by NIH Accelerated Excellence in Translational Sciences (AXIS) grant number 2U54MD007598-07; and the University of California at Los Angeles (UCLA) Clinical and Translational Science Institute (CTSI), grant number UL1TR001881.


A total of nine patients developed major cardiovascular events. Patients with CHIP did not have a higher risk of major cardiovascular events, although patients with DNMT3A did have a higher risk , independent of other variables. We did not find that CHIP was associated with a greater risk of silent heart disease or cardiovascular events, although those affected by DNMT3a, analyzed independently, were associated with a greater number of cardiovascular events.Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the clonal expansion of hematopoietic stem cells carrying certain genes associated with an increased risk of hematological malignancies. Our study analyzes the influence of CHIP on the risk of heart disease and cardiovascular events in a population with chronic kidney disease (CKD). A total of 128 patients were prospectively followed up for 18 months to detect major cardiovascular events (MACE). To detect the presence of silent heart disease, troponin I, NT-Pro-BNP, and coronary calcification were measured. A massive sequencing was performed to detect CHIP. A total of 24.2% of the patients presented CHIP, including that which was only pathogenic. The most frequently affected gene was TET2 (21.1%). Using multivariate logistic regression analysis, the presence of CHIP was not related to coronary calcification (OR 0.387, 95% CI 0.142–1.058,  Chronic kidney disease (CKD) is an inflammatory disease with a high prevalence in all populations of the world, affecting around 9% of the adult population . AlthougClonal hematopoiesis of indeterminate potential (CHIP) is defined by the clonal expansion of hematopoietic stem cells carrying mutations in certain genes that are independently associated with increased risks of hematologic malignancies, atherosclerotic cardiovascular disease, worse heart failure outcomes, and all-cause mortality . It is kOur hypothesis was that, because it is an inflammatory disease related to aging, CKD may favor the appearance of these mutations in the transcriptional regulatory genes that define CHIP. These mutations may contribute to the pathogenesis of CKD-associated heart disease or could be useful as new CKD biomarkers. To test this hypothesis, we conducted a prospective study in a cohort of CKD patients without previously known cardiovascular diseases, analyzing the presence of subclinical heart disease  at the time of CHIP determination and the subsequent appearance of clinical heart disease.Patients with chronic kidney disease being monitored in nephrology clinics  were included between 1 September 2020 and 31 January 2021. They had to be older than 18 years old and interested in participating in the study, as well as willing to sign the informed consent. The exclusion criteria were previous cardiovascular pathology, atrial fibrillation, and previous diagnosis of any neoplasia . In total, 128 patients gave their consent to participate in the study. The study was approved by the Cantabria Clinical Research Ethics Committee (2020:326).® software , and the amount of calcium was expressed according to the system proposed by Agatston. Agatston’s classification is defined as follows: 0–100, 100–200, over 400, etc. To carry out the study, the total amount of coronary calcium and the amount observed in the anterior descending artery were evaluated.Coronary artery calcification obtained via computed tomography using 64/128 Optima (64) and Revolution EVO (128) detectors, GE Healthcare, USA, was measured with the prospective acquisition technique with heart rate monitoring. The amount of calcium in the coronary arteries was measured using SmartScore® Healthineers, Tarrytown, NY, USA); and vitamin D (25-OH-vitamin D) was analyzed via automated chemiluminescence in a Liaison XL® (DiaSorin). In addition, a frozen serum sample was kept at −80 °C for specific determinations . Troponin I (High Sensitivity Troponin I) and NT-pro BNP were performed using automated immunoassay on an Atellica® IM .Together with the collection of anthropometric and clinical data, a blood sample was taken the same day in a standardized manner, in which creatinine (estimated glomerular filtration rate using the CKD-EPI equation), calcium, phosphorus, PTH, and LDL cholesterol were immediately analyzed; HDL cholesterol was analyzed via standardized, automated analysis in an Atellica CH Solution equipment and an Atellica IM Solution equipment  in QIAcube (Qiagen). DNA concentrations were obtained using the Qubit dsDNA HS Assay Kit  in a Qubit 4 Fluorimeter (Life Technologies). The massive sequencing study was performed with Ion Chef System On-Demand technology using the Ion AmpliSeqTM Kit for Chef DL8 (Ion Torrent Cat. No. A29024). The panel design was performed at AmpliSeq.com for the TET2, DNMT3A, and MPL genes, with a total number of 161 amplicons, with full coverage of the coding region +/- 10 intronic bases to cover splicing regions. The in silico coverage was 100%, although a real coverage of 97% was obtained. In the first step, gene libraries were prepared using 2 pools of primers, 20 amplification cycles, and an alignment time and an extension of 4 min. In the second step, sequencing via synthesis was carried out on a 520 chip (Ion Torrent Cat. No. A27762); 32 patients with minimum coverage of 1000× and an analytical sensitivity of 4% were analyzed on each chip. Torrent SuiteTM software was used for data analysis, and Varsome software was used to record pathogenicity, which was assessed according to the ACMG criteria . Patients were prospectively followed up until 30 June 2022. A major cardiovascular event (MACE) was defined if the patients presented an acute myocardial infarction requiring PTCA/stent, coronary bypass, stroke, transient ischemic attack, peripheral arterial ischemia requiring PTCA/stent, amputations, limb bypass, arterial thrombosis, cardiogenic shock, or cardiac arrest.Our main objective was to assess the relationship between the presence of mutations in the transcriptional regulatory genes that define CHIP and the occurrence of cardiovascular (CV) events. The secondary objective was to analyze the relationship between the presence of CHIP and the presence of subclinical heart disease assessed via imaging  or laboratory tests (Troponin I and NT-proBNP determination).Continuous variables were expressed as the mean and standard deviation. Categorical variables were described as relative frequencies. The characteristics of patients with and without CHIP and with and without a global coronary calcification value measured by an Agatston score of ≥400 were compared by comparison of means using a t-test for continuous variables and Chi-square for categorical variables. Variables independently related to an Agatston global coronary calcification score of ≥400 were analyzed using multivariate logistic regression. Variables related to the development of MACE were analyzed using Kaplan–Meier analysis and univariate and multivariate Cox regression. Statistical analysis was performed using SPSS version 15.0 .The presence of CHIP was determined in 128 patients, with a mean age of 61 ± 7 years, and a coronary calcification study was performed on 127 due to the death of a participant before carrying out the radiological study. p = 0.064), while diabetes mellitus  and phosphorus  were independently associated with the risk of coronary calcification being ≥400 . Performing the same analysis and substituting CHIP for the presence of TET2 mutations, this variable was also not independently related .The presence of CHIP was associated with a lower risk of global and anterior descending coronary calcification and was not associated with other variables related to silent heart disease, such as troponin and NT-ProBNP. The prevalence of CHIP was not higher in older patients with a more advanced degree of CKD or in patients with stage 5 CKD, and it was lower in diabetics. The relationship between the different variables and global coronary calcification, established as an Agatston value of ≥400, is described in p = 0.015) of those with CHIP, coronary calcifications, stage 5 CKD, and phosphorus.In our group of patients with CKD without previous cardiovascular pathology, we did not find a relationship between the presence of CHIP and silent heart disease detected as coronary calcification or blood markers associated with heart disease, such as troponin I and NT-ProBNP. In fact, in the univariate study, CHIP seemed to protect against the risk of coronary calcification. This finding was due to the lower prevalence of diabetics in our group of patients with CHIP, demonstrating that the protective effect of CHIP  on the risk of coronary calcification disappeared after the multivariate study. A previous study in the general population observed that there was indeed a relationship between the presence of CHIP and an increased risk of coronary calcification. Jaiswal et al. demonstrated that patients with CHIP without prior atherothrombotic disease in a Bioimage study had a more than threefold increased risk of having a coronary calcification value greater than or equal to 615, regardless of age, sex, the presence of diabetes or hypertension, total cholesterol, HDL, or smoking . AlthougThe markers selected to detect silent heart disease have been related to the appearance of subsequent cardiovascular events both in the general population and in patients with CKD. The presence of coronary calcification measured with the Agatston scale is known to be related to overall mortality and cardiovascular events through prospective studies that have included thousands of patients ,16, offeIn their initial study including 17,182 patients, Jaiswal et al. found that the presence of somatic mutations of genes associated with hematological malignancies in peripheral blood cells was associated with an increased risk of overall mortality , ischemic heart disease , and ischemic stroke , subsequently corroborating this relationship between CHIP and greater atherogenesis in animal models . Unlike In our study, we did not find that the prevalence of CHIP was related to worse renal function (either as a continuous variable or for each stage of CKD or in stage 5 patients) or with other variables that could condition more chronic inflammation or a worse environment that would justify changes in DNA, such as smoking or changes in phospho-calcium metabolism. A previous study observed that patients with CHIP were associated with a lower cystatin-estimated glomerular filtration rate but, as shown in our study, not with the creatinine-estimated glomerular filtration rate . In the Our study has various limitations, the most important being the smaller sample size and the shorter follow-up time compared with other studies ,11,12,25In conclusion, in our CKD population without previous cardiovascular pathology, we did not find a relationship between silent cardiac pathology and the prevalence of CHIP mutations. However, we selectively found that mutations in one of the CHIP genes, DNMT3A, were associated with a high risk of major cardiovascular events independently of other well-established variables, while the rest of the genes were not. This differential effect of DNMT3A on the cardiovascular risk of CKD patients and the influence of renal dysfunction on the appearance of CHIP should be studied in future studies.