Patients had significantly lower vitamin D, calcium levels, and z-scores. Lumbar Cobb angle was significantly correlated with the z-score , with right foot fifth metatarsal load , center of pressure CoPx , CoP displacement  and 90% confidence ellipse area . (4) Conclusions: In our study including female adolescents with idiopathic S shaped moderate scoliosis, plantar pressure and stabilometric parameters were influenced by the main scoliotic curve.(1) Background: Adolescent idiopathic scoliosis (AIS) can be associated with vitamin D deficiency and osteopenia. Plantar pressure and stabilometry offer important information about posture. The objectives of our study were to compare static plantar pressure and stabilometric parameters, serum 25-OH-vitamin D3 and calcium levels, and bone mineral densitometry expressed as z-score in patients with moderate AIS and healthy subjects. (2) Methods: 32 female adolescents  and 32 gender and age-matched controls performed: static plantar pressure, stabilometry, serum 25-OH-vitamin D3 and calcium levels, and dual X-ray absorptiometry scans of the spine. (3) Results: In scoliosis patients, significant differences were recorded between right and left foot for total foot, first and fifth metatarsal, and heel loadings. Stabilometry showed a poorer postural control when compared to healthy subjects ( Scoliosis is a three-dimensional spinal abnormality. The idiopathic one is the most prevalent type in adolescents (80–90%) . For thaNumerous factors are thought to be involved in the etiology of scoliosis. There seems to be a connection between the appearance of scoliosis and child growth and development, hormone release, posture, and physical activity ,6,7,8,9.Vitamin D is a major factor involved in the bone growth; if deficient during first years of life, it is associated with poor skeletal mineralization, leading to a wide variety of skeletal deformities. While some studies have reported that vitamin D deficiency is frequent in patients with AIS, the modality by which vitamin D influences scoliosis is not established .There are studies that also revealed an important association between scoliosis and osteopenia ,17,18,19Plantar pressure measurement provides useful objective information for physicians; it is an important tool to determine the treatment option for an enhanced clinical outcome . VariousThere are only few studies that emphasized the relationship between modified static plantar pressure parameters and scoliosis; most investigations highlighted the importance of center of pressure trajectory in gait ,31,32,33The hypothesis of our study was the fact that scoliosis can influence the static plantar pressure and stabilometric parameters and may be related with vitamin D deficiency and low bone mineral density.The main aim of the current research was to compare the static plantar pressure and stabilometric parameters between female adolescents with idiopathic scoliosis and healthy subjects. Another objective was to analyze the serum levels of 25-OH-vitamin D3 and calcium, and bone mineral density in the two groups. To our knowledge there is no study that has assessed the static plantar pressure and stabilometric parameters, bone mass index, and vitamin D altogether in adolescent idiopathic scoliosis.The current research was achieved in accordance with the Declaration of Helsinki, being approved by Institutional Ethics Committee . Participation in the study was voluntary. All the participants’ parents gave their written informed consent.The parents of 36 children were requested to take part in the study. The children were patients of the Pediatric Surgery Department, “Louis Turcanu” Emergency Children’s Hospital Timișoara, România. The general practitioners mainly addressed the children to the hospital. However, there were some cases in which the parents came to the hospital as they had noticed the poor posture of their children.Inclusion criteria were: post-menarcheal adolescent girls; idiopathic S shaped scoliosis , with a lumbar Cobb angle between 25–45°. Exclusion criteria were: congenital or other musculoskeletal disorders, neurologic, endocrine, infectious, malignant, and psychiatric diseases. Four patients met the exclusion criteria: clubfoot deformity (2); femur bone cyst (1); hypoparathyroidism (1). We enrolled 32 patients. Thirty-two gender and age-matched healthy controls joined the study after finding out information from posters placed at city schools. The controls were healthy adolescents; they did not suffer from any musculoskeletal disorder. Before being considered controls, they needed to have undergone clinical examination. The medical history was also recorded. The students’ parents were asked if they were willing to take part in the study. The permission to place information posters was obtained from the schools.A sample size analysis was performed using the G*Power 3.1.9.2 software . For a power of 80% beta = 0.2, alpha = 0.05 and an estimated effect size of 0.7, the number of subjects for each group should be at least 26 .The study was conducted between January 14, 2019 and September 30, 2019.Patient and control features were collected. The participants were assessed at “Louis Turcanu” Emergency Children’s Hospital Timișoara, România. The patients were assessed clinically by the same investigator. The protocol of our research implied one assessment for the study group and controls. The following determinations were performed: static plantar pressure analysis and stabilometric analysis, X-rays of the thoracic and lumbar spine (standing position) with the measurement of Cobb angle by a single experienced investigator, serum 25-hydroxyvitamin D level, and bone mineral density measurement. The 2016 SOSORT (Scientific Society on Scoliosis Orthopedic and Rehabilitation Treatment) guidelines define mild scoliosis by a Cobb angle less than 20°; moderate scoliosis is characterized by a Cobb angle between 21° and 35° [For the static plantar pressure assessment, we used a capacitive system . It records data of weight distribution, barycenter, and stabilometry ,37. The The device registered the plantar pressure distribution on first and fifth metatarsal heads, and calcaneus ,38. For Y) and latero–lateral (CoPX) axes [Y axis and a right deviation on the CoPX axis signify a positive value. The system also determined the absolute mean CoP displacement from the ideal position [2) of the ellipse containing all the center of gravity points measured and transferred on a system of Cartesian axes; the confidence level is 90%. Maximum CoP speed (expressed in millimeters per second) means the average center of gravity shifting maximum speed.Body center of pressure deviation from hypothetical framework was measured on anterior–posterior (CoPPX) axes ,39,41. Aposition . We analposition . The CoPThe blood samples were collected by the same investigator. Venous blood was sampled for determining serum calcium and serum 25-OH-vitamin D3 (25OHD) in the morning, after an 8-hour fasting. Serum calcium was measured by automated biochemistry analyzer Cobasc-501 . The normal values for serum total calcium were between 8.8 and 10.8 mg/dL (in 2–12-year old children) and between 8.4 and 10.5mg/dL (in 12–18-year old children). The serum 25OHD was determined using the chemiluminescence method . A serum 25OHD level less than 20 ng/mL was defined as Vitamin D deficiency. Vitamin D deficiency was considered severe (25OHD < 5 ng/mL), moderate (25OHD < 10 ng/mL) or mild (25OHD < 20 ng/mL) .2), expressed as z-scores. According to the Official Pediatric Positions of the International Society for Clinical Densitometry, low bone mineral mass or bone mineral density is the favored nomenclature for pediatric DXA reports when BMC or areal BMD z-scores are equal or below −2.0 [Dual X-ray absorptiometry (DXA) is frequently used to define osteoporosis . All DXAlow −2.0 ,47. The p < 0.05 for all tests. The statistical analysis was performed with the MedCalc Statistical Software version 19.1 .The Shapiro-Wilk test was used for testing data‘s normality. The homogeneity of variances between groups was tested with Levene’s test. Demographic data  between patients and controls were compared with t-tests and Fisher exact tests. Mann–Whitney tests and t-tests for independent measures were performed to assess the differences between patients and control groups, for non-normal distributed data  and normal distributed data, respectively . The Wilcoxon test was used to investigate the differences between limbs for the plantar pressure load distribution. The relationship between levels of calcium, vitamin D, Cobb’s angle, and stabilometric parameters was analyzed with the Spearman‘s rank correlation coefficient. The significance level was set at p > 0.05)  .The serum vitamin D and calcium levels, DXA z-scores, and Cobb’s angle values are presented in The results of plantar pressure analysis are presented in p < 0.0001). The load on the first metatarsal was significantly decreased, while on the fifth metatarsal it was significantly increased in patients in comparison to the controls for both right and left feet. The adolescents with scoliosis had a significantly lower heel load on the right foot. There were no significant differences in left heel loadings between patients and controls (p = 0.38).When analyzing the total foot load between children with scoliosis and controls, we found a significantly higher load on the left foot and a significantly lower load on the right foot in the patient group (The stabilometric data are presented in p = 0.02), the right foot MT5 load , COPX , COP displacement  and 90% confidence ellipse area .The lumbar Cobb angle (the main scoliotic curve) was significantly correlated with the following: the z-score . This overload on the left foot corresponds to the left convex lumbar curve. Regarding the comparison of the three sites of weight distribution between the right and the left foot we noticed a higher load on the first and fifth metatarsal heads on the right side, with an increased load on the left calcaneus. When compared to gender and age-matched healthy adolescents, important differences in weight loadings were reported for both limbs. These differences involved the total foot load and the three weight distribution sites, except for the left heel load.Our research was focused on the evaluation of the static plantar pressure and stabilometry in female adolescents diagnosed with idiopathic S shaped moderate scoliosis . Our patients had an increased total foot load on the left limb in comparison to the right one  carried out on thirty 10-year old girls diagnosed with right-side idiopathic thoracic mild degree scoliosis noticed that a higher pressure was imposed on the hallux and convex front part of the foot . The stuIn the present research we also studied the deviation of the barycenter on the anterior–posterior and latero–lateral axes. Adolescents diagnosed with S shaped scoliosis with the main lumbar curve had a higher deviation anteriorly and to the left. This finding suggests an elevated support on the left forefoot; the leg corresponds to the lumbar convex curve. The CoP displacement represents a measurement of the body’s neuromuscular need to preserve its vertical stability. Previous studies indicated that the trajectory of CoP is an important biomechanical tool for assessing the postural stability ,49,50. LIn the current study we also compared the serum vitamin D and calcium levels, as well as the bone mineral density between female adolescents with scoliosis and healthy controls. Some studies noticed a possible association between vitamin D levels and scoliosis ,52. BaliWe found that the lumbar Cobb angle was significantly correlated with the z-score; a higher lumbar scoliosis angle correlates with a lower DXA score. The results are related to Warren et al. (2005) ; they coThe adolescents included in our study received the instruction for wearing thoracolumbosacral orthosis for 23 h per day. They also started the rehabilitation program and were directed to the pediatric department for the treatment of vitamin D deficiency. Previous researches have reported beneficial effects of conservative treatment in AIS and other musculoskeletal diseases, with long-term consequences on the quality of life ,59,60,61The plantar pressure analysis should contribute to a patient tailored exercise program. This recommendation will have implications for both research and clinical practice. The corrections of the posture and body alignment, as well as the plantar pressure equalization under visual control, were the main targets of the rehabilitation indicated for our patients. The implication for research can be represented by the prognostic value of the plantar pressure in the evolution of scoliosis.The current research has some limitations. We included a relatively small number of female adolescents with moderate scoliosis, with main left convex lumbar curve. Additional studies are required to analyze the effects of all types of scoliosis on plantar pressure in a balanced gender distribution.In our study including female adolescents diagnosed with idiopathic S shaped moderate scoliosis, the static plantar pressure and stabilometric parameters were influenced by the main scoliotic curve. Important differences in weight distribution and stability were recorded in comparison to healthy controls. These data, associated with the vitamin D deficiency and the high percentage of low bone mineral density, may contribute to the follow-up of body alignment of patients with scoliosis undergoing conservative treatment.


Baroreflexes and peripheral chemoreflexes control efferent autonomic activity making these reflexes treatment targets for arterial hypertension. The literature on their interaction is controversial, with suggestions that their individual and collective influence on blood pressure and heart rate regulation is variable. Therefore, we applied a study design that allows the elucidation of individual baroreflex–chemoreflex interactions.We studied nine healthy young men who breathed either normal air (normoxia) or an air–nitrogen–carbon dioxide mixture with decreased oxygen content (hypoxia) for 90 min, with randomization to condition, followed by a 30-min recovery period and then exposure to the other condition for 90 min. Multiple intravenous phenylephrine bolus doses were applied per condition to determine phenylephrine pressor sensitivity as an estimate of baroreflex blood pressure buffering and cardiovagal baroreflex sensitivity (BRS).p < 0.001), raised heart rate from 62.9 ± 2.1 to 76.0 ± 3.6 bpm (p < 0.001), but did not change systolic blood pressure (p = 0.182). Of the nine subjects, six had significantly lower BRS in hypoxia (p < 0.05), two showed a significantly decreased pressor response, and three showed a significantly increased pressor response to phenylephrine in hypoxia, likely through reduced baroreflex buffering (p < 0.05). On average, hypoxia decreased BRS by 6.4 ± 0.9 ms/mmHg  but did not change the phenylephrine pressor response (p = 0.878).Hypoxia reduced arterial oxygen saturation from 98.1 ± 0.4 to 81.0 ± 0.4% (We applied an approach to assess individual baroreflex–chemoreflex interactions in human subjects. A subgroup exhibited significant impairments in baroreflex blood pressure buffering and BRS with peripheral chemoreflex activation. The methodology may have utility in elucidating individual pathophysiology and in targeting treatments modulating baroreflex or chemoreflex function. Arterial baroreflexes and peripheral chemoreflexes have been implicated in the pathogenesis of arterial hypertension and identified as treatment targets . Both or2 were enrolled in the study  concentration, breathing frequency, tidal volume, minute ventilation , and an electrocardiogram recording . An antecubital venous catheter was set in place for drug administration. Baseline data were recorded for 10 min prior to starting the randomized protocol , upper arm blood pressure, arterial oxygen saturation, end-tidal carbon dioxide . Bolus flow rates were 5 ml/s for phenylephrine and 2.5 ml/s for the saline bolus, resulting in administration durations of 4.1–4.4 s. Following determination of the appropriate dose, we obtained repeated baroreflex measurements using the predetermined phenylephrine dose, with the subjects exposed to either the normoxic or hypoxic condition for 90 min, with randomization to one or the other condition and single blinding (subjects). Following the first exposure, subjects were allowed to recover for 30 min during which time they could stand up and empty their bladder, with the aim to minimize any carry-over effects from being supine or hypoxic, before being exposed for 90 min to the other condition. In the first 10 min of each exposure, inspiratory oxygen was slowly lowered to attain hypoxia or kept constant for normoxia testing. Thereafter, 20 phenylephrine bolus doses were applied, one every 4 min. The study protocol is illustrated in Fig. We utilized a hypoxic gas mixture containing nitrogen, oxygen, and carbon dioxide. Compressed air and nitrogen were mixed in a blender . Carbon dioxide was added separately in order to maintain normocapnia. Gas flows were controlled using flowmeters and buffered in a Douglas bag from which the subject could inhale without resistance or pressure via a face mask. For normocapnic hypoxia, the target was 80% arterial oxygen saturation, achieved by manual adjustment. To keep subjects blind to the condition, the same toolset was used to apply compressed air during both the hypoxia and normoxia procedures.r > 0.5 [Cardiovagal BRS was assessed as the slope of the regression line between each systolic blood pressure (SBP) value and the following RR interval during the phenylephrine-induced pressure rise without an offset , 26. Ther > 0.5 .Larger increases in blood pressure after the administration of a defined phenylephrine bolus denote reduced the baroreflex buffering capacity. We determined this phenylephrine sensitivity as rise in SBP (ΔSBP) from the baseline before each bolus to the SBP maximum after bolus application . BaselinTwo parameters that characterize baroreflex function were assessed, namely, cardiovagal baroreflex sensitivity (BRS) and baroreflex buffering capacity. For both measurements we utilized the increase in finger blood pressure after the administration of each phenylephrine bolus. This pharmacological approach ensures greater independence from ventilatory influences on baroreflex function than methods based on spontaneous variations in hemodynamic variables. The investigator was blinded to the condition, i.e., normoxia or hypoxia.t tests. For group comparisons, we used medians of up to 20 boli per condition in order to minimize the effect of possible outliers and analyzed these via the paired t test. Significance was assumed at p < 0.05. If not otherwise indicated, all values were expressed as the mean ± SE. Power for BRS and change in SBP were calculated post hoc by means of the statistical analysis tool G*Power 3.1. We estimated the number of bolus repetitions needed to estimate individual effects between normoxia and hypoxia with receiver operating characteristic curve analysis performed with SPSS V.21 software . All other statistical analyses were done using Graphpad Prism 7.04 Individual BRS and phenylephrine responses were analyzed using unpaired p < 0.001), and ETCO2 remained at baseline values . Respiratory rate  did not change. During hypoxia, tidal volume increased from 0.77 ± 0.04 to 0.95 ± 0.07 l (p = 0.002), and minute ventilation increased from 11.86 ± 0.51 to 13.95 ± 0.49 l/min (p = 0.036); the mean respiratory chemoreflex response (ΔVe/SpO2) was 11.79 ± 4.80 (l/min)/%.All subjects tolerated baroreflex testing in both normoxia and hypoxia, and no adverse events were observed. Arterial oxygen saturation was 98.1 ± 0.4% during normoxia and 81.0 ± 0.4% during hypoxia (p < 0.001). Figure Blood pressure was 133 ± 3/76 ± 2 mmHg during normoxia and 135 ± 3/76 ± 2 mmHg during hypoxia. During the experiment systolic blood pressure ranged from 122 to 154 mmHg during normoxia and from 121 to 148 mmHg during hypoxia. Heart rate increased from 63 ± 2 bpm during normoxia to 76 ± 4 bpm during hypoxia , as shown in Fig. Five subjects showed a significant change in phenylephrine responsiveness with hypoxic peripheral chemoreflex stimulation Table , with phThe optimal number of repetitions for individual BRS changes was ten boli with a sensitivity of 0.96. This is derived from our heathy subjects with an effect size of 6 ± 3 ms/mmHg (difference in BRS between normoxia and hypoxia).Overall, we did not observe a linear correlation between changes in BRS or phenylephrine responsiveness and respiratory parameters . Individual respiratory parameters varied, as shown in Hemodynamic reactions were stable over time during normoxia and hypoxia Fig. . The seqWe could not find a correlation between changes in cardiac BRS and baroreflex buffering capacity Fig. .Fig. 4CoIn the study reported here, we applied a novel approach to elucidate individual baroreflex–chemoreflex interactions using repeated administrations of a standardized phenylephrine bolus during normoxia and hypoxia. While individual and group cardiovagal BRS responses to hypoxic peripheral chemoreceptor stimulation showed similar qualitative responses, individual responses exhibited substantial quantitative variability. Strikingly, individual phenylephrine responsiveness revealed qualitatively different responses, possibly resulting from changes in vascular sensitivity, in baroreflex blood pressure buffering, or in both mechanisms combined . Our finThe highly standardized assessment of baroreflex function using phenylephrine doses applied through an automated injector is a particular strength of our study. In previous studies, vasoactive drugs for baroreflex testing as well as subsequent saline flushes were manually applied , 29. TheWe observed complex changes in baroreflex regulation with hypoxic peripheral chemoreflex stimulation. Average blood pressure was unchanged while the heart rate increased substantially. The baroreflex heart rate/pressure relationship was shifted towards higher heart rates and cardiovagal BRS was reduced. Increases in heart rate and maintained or increased blood pressure with peripheral chemoreflex stimulation have been previously described; in these studies sympathetic and parasympathetic baroreflex gains , 30, 31 Baroreflex regulation of sympathetic traffic to the vasculature is particularly relevant for blood pressure control. However, direct sympathetic nerve recordings are available in only a few medical centers and the tests often fail for technical reasons. As an alternative, the primary function of the baroreflex, which is to buffer blood pressure changes, can be assessed by applying vasoactive drugs before and during complete interruption of the baroreflex arc through ganglionic blockade ; the sen2, which affects cardiovascular control, was kept constant during our study. While we applied hypoxia in a steady-state fashion, transient hypoxia has been proposed to provide a more selective peripheral chemoreceptor stimulus [The major limitation of our study is that findings in healthy subjects cannot be simply extrapolated to patients with arterial hypertension, particularly those considered for device-based treatments targeting the baroreflex or peripheral chemoreceptors. The fact that we did not assess sympathetic nerve activity is another potential limitation. Moreover, we determined an estimate of baroreflex blood pressure buffering rather than making a comparison phenylephrine responses before and after ganglionic blockade for the reasons mentioned above. While much of the interindividual variability in phenylephrine responsiveness can be explained by baroreflex blood pressure buffering, hypoxia-induced changes in vascular alpha-adrenoreceptor responsiveness may have confounded the analysis. However, data from human  and animstimulus . Finallystimulus —althoughstimulus .Despite these limitations, our study suggests that interindividual variability of baroreflex and chemoreflex regulation is so high that treatments modulating these mechanisms are exceedingly unlikely to be equally effective in all patients. In recent years there has been a thrust towards precision medicine . Usually


Chronic kidney disease (CKD) is a serious complication of diabetes associated with adverse outcomes of renal failure, cardiovascular disease and mortality. Despite this, data regarding the burden and awareness of CKD among adults with diabetes in Sub-Saharan Africa countries are lacking. The aim of this study was, therefore to determine the prevalence and awareness of CKD among diabetic outpatients attending a hospital in Northeast Ethiopia.2 and/or albuminuria. Awareness was defined as a positive response to “Has a doctor or other health care professional ever told you that you had kidney disease?”We conducted a cross-sectional study on 323 diabetic adults at the diabetes clinic of a hospital in Northeast Ethiopia, from February 1 to July 30, 2016. Each patient provided a blood sample for serum creatinine and urine for albuminuria. Glomerular filtration rate (eGFR) was estimated using the Modification of Diet in Renal Disease (MDRD) equation. CKD was defined as eGFR < 60 ml/min/1.73 m2 and 58 (18.0%) had albuminuria. In patients with eGFR < 60 ml/min/1.73m2 (stage 3–5 CKD), serum creatinine was abnormal (> 1.5 mg/dl) in 23.5% and albuminuria was absent in 31.8%. Of the patients with CKD, only 10.6% of them were aware of their CKD. The proportion of patients who were aware of their disease increased with worsening of CKD stages, from 3.4% of with stage 1 to 75.0% with stage 4. Awareness for all individuals with advanced stages of CKD was only 11.9%. Having albuminuria, high serum creatinine, a family history of kidney disease and being obese were significantly associated with CKD awareness.Of the 323 patients, 85 (26.3%) had Stage 1–5 CKD, 42 (13.0%) had eGFR < 60 ml/min/1.73mA high prevalence but low awareness of CKD was found in diabetic outpatients attending our clinic in Northeast Ethiopia. Our results highlight the need for more diagnostic strategies for CKD screening among diabetic adults and primary care education on the impact of detecting CKD in the early stage to prevent adverse outcomes and improve diabetes care. Chronic Kidney Disease (CKD) is a global public health problem associated with an increased risk of end stage renal disease (ESRD), cardiovascular disease (CVD) and premature mortality . It is eCKD is a well recognised and serious complication of diabetes and diabetes is one of the most common causes of CKD, with up to 44% of patients with CKD affected by diabetes , 6. It iAlthough CKD is common in diabetes and caries a high risk of adverse outcomes, it is often unrecognized; up to 77% of patients receiving diabetes management may have undiagnosed CKD , 12. In Despite existing evidence suggesting that CKD is a substantial health burden among diabetic patients on the African continent , data reThis cross-sectional study was conducted from February 1 to July 30, 2016 at the outpatient diabetes clinic of Dessie Referral Hospital (DRH), Northeast Ethiopia. The Hospital is found in Dessie town of Amhara regional state and serves as a referral center for South Wollo and surrounding zones. It delivers diagnostic, therapeutic and monitoring services to all diabetic patients and also tertiary diabetes patient care. Adult patients (aged ≥18 years) already diagnosed with diabetes mellitus attending the follow-up diabetes clinic of the hospital during the study period were included in the study. Diabetic patients who were hospitalized, pregnant (woman), with acute illness (fever), underwent treatment with dialysis and not fasting were excluded from the study. The study protocol was approved by the Institutional Review Board and Research Ethics Committee of Wollo University, College of Medicine and Health Sciences. An informed written consent was obtained from all the participants enrolled.The sample size calculation was based on an estimated prevalence of CKD of 50% in that population, with a confidence level of 95 and 5% precision. A sample size of at least 317 individuals was needed to be representative of the 1800 individuals on diabetic follow-up in our site to obtain 80% power. Considering a 5% non-response rate, the sample size was increased to 333 patients. Estimates of CKD prevalence were obtained on the 323 participants. Systematic random sampling was used to select study participants.2), overweight (BMI 25–29.9 kg/m2) and obese (BMI ≥30 kg/m2). Blood pressure (BP) was measured using a manual mercury sphygmomanometer, with the patient remaining in a sitting position for at least 15 min before the first measurement. Each participant’s blood pressure was measured three times at an interval of at least 5 min, and then averaged to be recorded. Hypertension was defined as systolic BP ≥140 mmHg and/or diastolic BP ≥90 mmHg or current use of antihypertensive medication. Awareness of kidney disease was ascertained by asking the question “Have you ever been told that you had a kidney disease by a doctor or other health care professional?” Do not include kidney stones or bladder infection.Data on socio-demographic, medical and family history were collected by using a pre-tested structured interviewer administered questionnaire  using kits supplied by the manufacturer. Blood glucose was measured by enzymatic GOD-PAP method. Serum creatinine was measured using a Jaffe kinetic method, with calibration traceable to IDMS reference material. Fresh urine sample was collected from each participant to detect albuminuria using dipsticks . Presence of albuminuria (+ 1 or more) in urine was defined as albuminuria.2 and/or albuminuria on two occasions spaced by 2 weeks. Stages of CKD were classified according to the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF KDOQI) guideline as [2 with albuminuria; stage 2, eGFR of 60–89.9 ml/min/1.73 m2 with albuminuria, and stages 3, 4 and 5 as eGFR of 30–59.9, 15–29.9 and < 15 ml/min/1.73 m2, respectively. Stage 3 was further classified into 3A (45–59.9 ml/min/1.73 m2) and 3B (30–44.9 ml/min/1.73 m2) [The four-variable Modification of Diet in Renal Disease (MDRD) study equation was used to estimate glomerular filtration rate (eGFR) . CKD waseline as : stage 11.73 m2) .2) test or t-test as appropriate. Multivariable stepwise logistic regression was used to calculate adjusted odds ratios (OR) and the corresponding 95% confidence intervals (CI). P value < 0.05 was used to indicate statistical significance.Data were entered into “EpiData 3.1” software and analyzed using SPSS version 20.0 . Data were expressed as means ± standard deviation (SD) or percentage. Comparisons between groups were done by chi square  age of the participants was 44 ± 15.7 years (ranging from 18 to 77 years), and 168 (52.0%) were males. Of the participants, 173 (53.6%) were type 2 diabetics, 186 (57.6%) had duration of diabetes less than 5 years, and 62 (19.2%) had a family history of kidney disease. Mean BMI was 22.37 ± 3.94 Kg/m2 (stage 3–5 CKD). Only 23.5% of the participants with advanced stage CKD had SCr values > 1.5 mg/dl  [2 (stage 3–5 CKD). The prevalence of CKD was 46.9% in participants > 60 years old and 22.6% in those ≤60 years old (P < 0.001). CKD prevalence was lower in males (14.8%) than in females .The overall prevalence of CKD stage 1–5 was 26.3%  (Table P < 0.001), female gender (P < 0.001), decreased literacy (P = 0.017), low monthly income (P = 0.042), long duration of diabetes (P < 0.001), family history of kidney disease (P < 0.001), hypertension (P < 0.001), high systolic BP (P = 0.005), and poor glycemic control (P < 0.001). In the multivariable analysis, older age , gender , longer duration of diabetes , hypertension , a family history of kidney disease , and poor glycemic control  were significantly associated with CKD  was only 11.9%. For logistic regression, CKD awareness was used as the outcome variable. The covariates were age, sex, duration of diabetes, family history of kidney disease, obesity, serum creatinine and albuminuria. In multivariable analysis, having albuminuria , abnormal serum creatinine , and a family history of kidney disease  and being obese  were all statistically significantly associated with greater awareness , 13.0% was found in our studied diabetic outpatients using eGFR equations. This is in accordance with other studies that found a prevalence of 3.9–31% of eGFR MDRD < 60 ml/min/1.73 m2 [2; stages 3–5 CKD). This is in accordance with our previous study, reporting only 48.7% of diabetic outpatients with eGFR MDRD < 60 ml/min/1.73 m2 had abnormal serum creatinine values [2). In line with this a recent study found that approximately one half (46.6%) of diabetics with CKD had reduced eGFR without albuminuria [A high prevalence of advanced stages CKD  using sex, age, race and serum creatinine only. Because it is more accurate than the Cockcroft-Gault equation in CKD patients with diabetes [The 4-variable MDRD study equation was used for estimating GFR in this study, as recommended by the NKF KDOQI guidelines, as it generates a GFR estimation normalized to a standard body surface area  of the patients had a short duration of diabetes, and this may explain some of the differences with other studies. We used the MDRD study equation, the validation of which is lacking among Ethiopian diabetic adults. The CKD-EPI equation may be superior in estimating the GFR but the best eGFR equation for Ethiopian adults has not yet been determined. Finally, the measurement of serum creatinine was not standardized; this might influence the performance of eGFR equations.In conclusion, this study demonstrated a high prevalence and low awareness of CKD among diabetic adults attending the outpatient diabetes clinic of Northeast Ethiopia. Current screening methods, serum creatinine or urine dipstick for albuminuria, underestimates the presence of clinically significant CKD in our diabetic outpatient. More diagnostic strategies for CKD screening among diabetic adults and primary care education on the impact of detecting CKD in the early stage are needed to prevent adverse outcomes and improve diabetes care.Additional file 1:. Questionnaire. English version of the questionnaire used during the study.


Higher values of ambulatory blood pressure monitoring parameters are associated with greater increases in diastolic blood pressure during isometric handgrip exercise. Diastolic blood pressure elevations during the handgrip test are also correlated, in order to diminished heart rate variability parameters attributable to parasympathetic dysfunction highlighting the pivotal role of sympathetic overactivity in evolving handgrip test results. Our study provides further evidence on the inverse association between handgrip test abnormality and hypertension in diabetic patients.Cardiovascular autonomic neuropathy (CAN) is a common complication of diabetes mellitus. Cardiovascular reflex tests (CARTs) are the gold standard in the diagnosis of CAN, but the handgrip test is no longer recommended to be performed. Previously, the inverse association between the presence of hypertension and handgrip test abnormality was demonstrated and hypertension as major cause for excessive diastolic blood pressure rise during handgrip testing in diabetic individuals proposed. The aim of the present study is to describe more precisely the association between handgrip test and hypertension by performing ambulatory blood pressure monitoring (ABPM) among diabetic patients. A more comprehensive evaluation of the relationship between cardiovascular autonomic function, hypertension and the handgrip test was targeted using heart rate variability (HRV) analysis. Our study involved 163 patients with diabetes. Cardiovascular autonomic neuropathy was assessed by the CARTs and sustained handgrip test was performed. All patients underwent ABPM and HRV analysis well. CAN was diagnosed in 69 patients. Significant associations were found between the diastolic blood pressure increase in response to handgrip exercise and the 24-h (rho = 0.245,  Cardiovascular autonomic neuropathy (CAN) is a common complication of diabetes mellitus  and it hConsequently, early and correct diagnosis of CAN cannot be overstated. Standard cardiovascular reflex rests (CARTs) are the most widely used in clinical practice. CARTs are safe, non-invasive, easy-to-perform tests and considered gold standard of CAN assessment . HistoriIn our recent study involving over 350 patients with diabetes, we proved that sustained handgrip test has a poor sensitivity and specificity against the diagnosis of confirmed CAN and no associations with results of other CARTs could be demonstrated. Therefore, we provided evidence that the isometric handgrip test should no longer be applied for the evaluation of sympathetic dysfunction during cardiovascular autonomic neuropathy testing in diabetic patients. Variables confounding handgrip test results proved to be the presence of hypertension and the initial diastolic blood pressure values during handgrip exercise. More precisely, the presence of hypertension and handgrip test abnormality were inversely and independently associated [Recommendations agree that ambulatory blood pressure monitoring (ABPM) is essential for the diagnosis of white-coat-, nocturnal- and masked hypertension  and dataSustained isometric handgrip test was originally considered a test to detect diabetic cardiovascular sympathetic neuropathy. In contrast, the inverse association between the handgrip test abnormality and the presence of hypertension found lately  implies Based on the above mentioned data, the present study aims to characterize more precisely the association between handgrip test and hypertension via performing ambulatory blood pressure monitoring (ABPM) among diabetic patients. Besides, we addressed the question of the relationship between cardiovascular autonomic function, hypertension and the handgrip test using heart rate variability (HRV) analysis.The present cross-sectional study involved patients with type 1 and type 2 diabetes mellitus presenting at the Department of Internal Medicine and Oncology, Semmelweis University, Budapest, Hungary from March 2014 to 2017 December. Type 1 and type 2 Diabetes Mellitus (DM) were established according to the WHO (1999) criteria . PatientThe study included 163 diabetic patients  who were eligible to participate. Eligible subjects were asked to avoid strenuous physical exercise, caffeine beverages, tobacco products and alcohol in the 12 h prior to cardiovascular autonomic assessment. Patients on antihypertensive agents that might confound outcomes of CARTs based on heart rate changes were instructed to omit interfering medication, particularly beta-receptor blockers and non-dihydropyridine-type calcium channel blockers in the 24 h interval before cardiovascular autonomic testing. Patients were requested to follow their normal daily life avoiding any stressful condition and consume their normal usual diet in the days before, and during, testing.All participants gave informed consent and the study protocol was approved by the local ethics committee. The datasets generated and analyzed during the current study are not publicly available due to its sensitive nature but are available from the corresponding author on reasonable request.2). Measurements of blood pressure (BP) values at rest and during the manoeuvres were performed using an OMRON M3  automatic upper hand-cuff sphygmomanometer. Office BP values were obtained after a minimal period of 5 min resting state and the average of three seated BP measurements was calculated. Cardiovascular autonomic function was assessed by the 5 standard CARTs using Cardiosys 12.1 diagnostic station and Cardiosys-A01 software . All tests were performed in a calm room according to standardised protocol [Data on age, glycaemic control (HbA1c), diabetes mellitus (DM) duration, antidiabetic and antihypertensive medication were acquired. Weight and height of eligible subjects were measured and body mass index (BMI) was calculated  mainly reflect parasympathetic function, whereas those based on BP changes to manoeuvres (handgrip test and orthostatic hypotension test) characterize sympathetic function. The results of the deep-breathing test were expressed as the difference of the highest heart rate during inspiration and the lowest heart rate during expiration . Valsalva ratio was assessed as the ratio of the longest RR interval after and the shortest RR interval during the manoeuvre. The results of the orthostatic hypotension test (OHT) were defined as the difference between the systolic BP obtained at rest in supine position and the lowest systolic BP after standing. 30:15 ratio during the lying-to-standing test was computed as the ratio of the RR intervals of the 30th and 15th (or nearby) sinus cycles following arising.During the handgrip test, measurements were obtained with the dominant hand. Subjects were comfortably seated without any armrest with the shoulders being adducted and neutrally rotated and with the elbow at 90° flexion and the forearm and wrist in a neutral position in accordance with the recommendations of the American Society of Hand Therapists’ . FirstlyTo evaluate parasympathetic neuropathy, age-related reference values for heart rate based tests were applied . The conAll patients underwent 24-h ambulatory blood pressure monitoring (ABPM) and ECG-recording as well. ABPM and HRV parameters were obtained using Meditech Cardiotens 1.34 device . PatientAs for HRV, frequency domain parameters were calculated from spectral analysis of normal-to-normal intervals using fast Fourier transformation; the low (LF: 0.04–0.15 Hz), the high (HF: 0.15–0.4 Hz) frequency components, the LF/HF ratio and total power (TP: 0.0–0.4 Hz) were evaluated.The Kolmogorov-Smirnov test for normality was accomplished on all variables. Normally distributed variables are expressed as mean ±SD while non-normally distributed data are described as median/geometric mean and interquartile range where appropriate. Categorical data are reported as n (%).2 test for categorical data were carried out based on the variable’s normality of distribution.For comparison between groups with and without handgrip test abnormality, Mann-Whitney U-test or paired t-test for continuous variables and χAssociations between the results of the handgrip test and ambulatory blood pressure monitoring (ABPM) parameters were analysed using Spearman’s rank correlation (rho) as neither diastolic blood pressure elevations during handgrip test nor ABPM parameters could be characterized by a stable normal distribution. Partial correlation was used for identifying significant correlations after adjustment for confounding variables.p < 0.05.All analyses were performed using SPSS 25 software . Statistical significance was defined as The present study involved a total of 163 patients: 20 (13.2%) with type 1 and 143 (87.7%) with type 2 diabetes; 148 (more than 90%) of our diabetic patients had coexistent hypertension as well. In 125 diabetic patients with hypertension, the diagnosis of hypertension was well known from past medical history and these patients were taking antihypertensive medication. We had 23 diabetic patients, in whom the diagnosis of hypertension was established upon ambulatory blood pressure monitoring. This way, 23 patients out of 38 (60.5%) diabetic patients with no previous hypertension in their medical history at baseline turned out to have hypertension upon ABPM studies. The main clinical characteristics of the study population are summarized in Diagnosis of confirmed CAN was proven in 42.3% of the patients. Abnormal results of the deep-breathing test, the Valsalva ratio, the 30/15 ratio, the handgrip and orthostatic hypotension test were present in 63.8%, 31.9%, 11%, 41.1% and 14.7%, respectively.normal handgrip test results had significantly higher 24-h mean systolic , higher daytime , night-time  mean systolic blood pressure values and 24-h mean diastolic  blood pressure values when compared to patients with abnormal handgrip test results. Similarly, patients with no handgrip test abnormality had higher systolic  and diastolic  blood pressure load and systolic  and diastolic  hyperbaric impacts than patients with abnormal handgrip test results. There was a trend towards diminished values of systolic and diastolic diurnal indices in the patient group with normal handgrip test result, but when compared to patients with abnormal handgrip test results, the difference did not reach statistical significance . In contrast, patient groups with normal and abnormal handgrip test result did not show any difference regarding age, BMI, diabetes duration, glycaemic control (HbA1c), presence of CAN and results of the other cardiovascular reflex tests. Among the HRV parameters, only total power (TP) (1717.2 vs. 1264.7 p = 0.047) was significantly reduced in patients with normal handgrip test result  compared to those with handgrip test abnormality , with daytime  and night-time  mean systolic blood pressure and diastolic  blood pressure values, with 24-h systolic blood pressure load  and with systolic  and diastolic (rho = 0.165. p = 0.047) hyperbaric impacts. As for HRV measures, HF  and TP , markers of parasympathetic function, showed weak but significant negative correlations to diastolic blood pressure response during isometric handgrip test (Correlations between ABPM and HRV measures and diastolic blood pressure elevations (in mmHg) during the handgrip test were also studied. Diastolic blood pressure response to handgrip test correlated significantly with 24-h mean systolic  parameters are associated with greater increases in diastolic blood pressure during isometric handgrip exercise. Furthermore, associations of handgrip outcomes with higher parameters of ABPM and with diminished HRV measures of parasympathetic autonomic function imply that results of the handgrip test are rather marker of sympathetic overactivity accompanying hypertension.CAN is an early and frequent complication of diabetes mellitus carrying a poor prognosis. Its prevalence reaches from 16.6% to as high as 65% in different diabetic patient populations depending on age, diabetes type and duration ,17. In tWe could not find any association between handgrip test abnormality and the presence of cardiovascular autonomic neuropathy. Handgrip test results did not show any relationships to the results of the deep-breathing test, the Valsalva ratio, the 30/15 ratio and the orthostatic hypotension test widely used for the assessment of sympathetic dysfunction, either. As a conclusion, the present study provides further evidence that diastolic blood pressure elevation in response to sustained handgrip exercise has no associations with other measures of CAN and should not be part of clinical CAN assessment. We could also reaffirm the inverse association between handgrip test abnormality and hypertension the latter previously proposed as the main confounder [ABPM is a safe, non-invasive method for the assessment of hypertension with many of its parameters being associated with cardiovascular morbidity and mortality . MoreoveIn the present study, patients with normal handgrip test result having (supra-) physiological blood pressure increase during short-time isometric handgrip exercise had significantly higher 24-h mean systolic, and diastolic blood pressure, systolic and diastolic systolic and diastolic blood pressure load and hyperbaric impacts, compared to diabetic patients with abnormal handgrip test result. Similar differences could be proven between groups in respect of daytime and night-time mean blood pressure values. Association with night-time blood pressures is of special clinical importance as they were reported as stronger predictor of cardiovascular events and mortality than daytime blood pressure .A correlation analysis was performed to get a more robust analysis of the association between diastolic blood pressure response to handgrip test and ABPM. Diastolic blood pressure elevations showed significant correlations with the 24-h, daytime and night-time mean systolic and 24-h diastolic blood pressures, with systolic blood pressure load and systolic and diastolic hyperbaric impacts. These correlations remained significant after adjustment for age, BMI and baseline diastolic blood pressure. Moreover, associations between diastolic blood pressure elevations and daytime and night-time diastolic blood pressure gained significance after adjustment. Therefore, our data provide evidence on independent, quantifiable correlations between the results of the isometric handgrip test and ABPM parameters in patients with diabetes mellitus. Interestingly, no significant correlations with diurnal indices could be proven though there was a trend toward diminished indices in the group with normal handgrip test result (>16 mmHg diastolic blood pressure response to handgrip exercise). On the one hand, this phenomenon could be a sample-size effect. On the other hand, diurnal indices might represent a different aspect of hypertension than mean blood pressure, blood pressure load or hyperbaric impacts given the fact that diurnal indices characterize the patients’ dipping status.According to our data, diastolic blood pressure response to isometric handgrip test correlated significantly negatively with HF and TP components of HRV analysis. HF and fluctuations in the overall variability (TP) are attributable to parasympathetic nervous system activity. These findings are in accordance with the theory that autonomic imbalance as a consequence of parasympathetic neuropathy and sympathetic overactivity may play a crucial role in the pathogenesis of hypertension in diabetic patients . In thisFindings in non-diabetic patients with hypertension imply that increased exercise vasoconstriction was a result of enhanced sympathetic activity and an impaired local vasodilatation during exercise in the contracting muscle . MicrovaSuch an easy-to-perform, quick and cost-effective test such as isometric handgrip test would be promising for screening of patients for hypertension and cardiovascular risk stratification. In the recent study performed among non-diabetic individuals , the firOur study has strengths and limitations. The strengths of the present study are the relatively large number of patients studied by ABPM and the adjustment for potential confounders, such as age, BMI and initial blood pressure values.One limitation could be the inclusion of both type 1 and 2 diabetic patients. However, neither literature data nor our previous studies revealed differences in features or pathogenesis of neuropathy between type 1 and type 2 diabetes. The mixed study population comprising both normo- and hypertensive individuals with diabetes could be considered as further limitation. Nevertheless, the fact that our study involved a broad spectrum of normotensive diabetic patients, patients with hypertension and patients in whom hypertension could only have been diagnosed via ABPM studies  should rather underline the general relevance of the associations. If our analysis were performed only in diabetic patients with hypertension, the associations between handgrip test and ABPM parameters might have been more pronounced. The high proportion of diabetic patients with treated hypertension might be considered as a limitation of the study. Therefore, the effect of antihypertensive medication cannot be excluded. Due to the cross-sectional design of our study, no conclusion on causal relationship between handgrip test result and hypertension could have been established.In summary, we could confirm the inverse association between handgrip test abnormality and ambulatory blood pressure parameters (ABPM) of hypertension. Higher values of ambulatory blood pressure monitoring (ABPM) parameters are associated with greater increases in diastolic blood pressure during isometric handgrip exercise and hence handgrip test abnormality will be very uncommon among these patients. Our results might involve the simple conclusion that the results of a diagnostic method based on measurement of blood pressure could not be independent from the presence of hypertension and as a consequence, handgrip test cannot be used to evaluate cardiovascular sympathetic neuropathy in diabetic patients. Moreover, associations of handgrip outcomes with higher parameters of ABPM and with reduced HRV measures of parasympathetic autonomic function suggest that diastolic blood pressure elevation during handgrip test should rather be considered a marker of sympathetic overactivity accompanying hypertension. The associations between diastolic blood pressure response to isometric handgrip test and measures of hypertension and target organ damage in this, and other studies, point to potential diagnostic and prognostic role of isometric handgrip test. Further prospective studies are warranted to clarify the potential role of diastolic hyper-responsiveness during the handgrip test as a screening tool for hypertension and cardiovascular risk prediction in diabetic and non-diabetic patients.


Acute early vascular complications are rare, but serious complications after kidney transplantation. They often result in graft loss. For this reason, shortening the diagnostic process is crucial. Currently, it is standard procedure to monitor renal graft perfusion using Doppler ultrasound (DU). With respect to acute vascular complications, the main disadvantage of this type of examination is its periodicity. It would be of great benefit if graft blood perfusion could be monitored continuously during the early postoperative period. It appears evident that a well-designed near infrared spectroscopy (NIRS) monitoring system could prove very useful during the early post-transplantation period. Its role in the immediate diagnosis of vascular complications could result in a significant increase in graft salvage, thus improving the patient’s overall quality of life and lowering morbidity and mortality for renal graft recipients. The aim of this study was to design, construct and test such a monitoring system.We designed a rough NIRS-based system prototype and prepared a two-stage laboratory experiment based on a laboratory pig model. In the first stage, a total of 10 animals were used to verify and optimize the technical aspects and functionality of the prototype sensor by testing it on the animal kidneys in-vivo. As a result of these tests, a more specific prototype was designed. During the second stage, we prepared a unique laboratory model of a pig kidney autotransplantation and tested the system for long-term functionality on a group of 20 animals. Overall sensitivity and specificity were calculated, and a final prototype was prepared and completed with its own analytic software and chassis.We designed and constructed a NIRS-based system for kidney graft perfusion monitoring. The measurement system provided reliable performance and 100% sensitivity when detecting acute diminished blood perfusion of the transplanted kidneys in laboratory conditions.The system appears to be a useful tool for diagnosing diminished blood perfusion of kidney transplants during the early postoperative period. However, further testing is still required. We believe that applying our method in current human transplantation medicine is feasible, and we are confident that our prototype is ready for human testing. Kidney transplantation is a standard medical procedure for patients suffering from chronic renal failure. In comparison with hemodialysis or peritoneal dialysis, it provides patients with better quality of life and a longer overall survival rate.Due to the shortage of suitable grafts worldwide, excellent long-term performance of the grafts is essential. During the early post-transplant period, sufficient and uninterrupted perfusion by arterial blood, as well as unhindered outflow of venous blood, is necessary for long-term graft survival and performance. Complications in this regard can cause delayed graft function, chronic graft dysfunction and, in extreme cases, primary a function and graft loss.Early vascular complications are among rare complications that can arise during the post-transplant period , 2. VascCurrently, it is standard procedure to monitor renal graft perfusion using non-invasive Doppler ultrasound –5, whichIt would be of great benefit if graft blood perfusion could be monitored continuously during the early postoperative period. In literature, two approaches are recommended when continuously monitoring kidney graft perfusion: measuring of electromagnetic waves with near-infrared spectroscopy (NIRS) –8 and moDoppler flow probe (DFP) uses ultrasound waves to monitor vascular flow and blood perfusion. However, this method has many disadvantages.In transplantation medicine NIRS has been used in experimental pediatric post-transplant monitoring, but only as a non-invasive method –8. This To our knowledge, there are currently no available invasive NIRS based measurement systems that would allow continuous monitoring of kidney graft perfusion during the postoperative period. It appears that a well-designed NIRS monitoring system could prove very useful in the early post-transplantation period. Its role in the immediate diagnosis of vascular complications could result in a significant increase in graft salvage, thus improving the patient’s overall quality of life and lowering morbidity and mortality rates.The aim of this experiment was to design, construct and test such a monitoring system.This study was reviewed and approved by the Ministry of Health of the Czech Republic—num. 29/2016.All legal and ethical requirements for this animal laboratory experiment have been met.The Institute for Clinical and Experimental Medicine, Prague, Czech Republic (IKEM), is in possession of an active license, which enables it to take part in experimental work on laboratory animals.All transplantations and procedures performed on laboratory animals were carried out in an operating theatre under the care of a trained anesthesiologist. After surgery, all animals were administered sufficient analgesic drugs. All animals were euthanized under full anesthesia at the end of the experiment.Based on previous studies , 11, 12 We prepared a rough NIRS measuring system prototype. The system was powered by its own energy source and was able to transmit wireless data directly to a secondary monitoring computer. The system was able to emit and monitor a broad spectrum of visible and near-infrared electromagnetic waves.We prepared a laboratory project based on a laboratory pig models. In-vivo testing was divided into two stages and three subgroups of laboratory animals.We chose laboratory pigs for our study for several reasons. The pig kidney is very similar to that of a human in anatomy and physiology , 14. TheKetamine+Azaperone+Atropine, full anesthesia was induced by i.v. bolus of Propofol+Fentanyl. The animal was placed on the operating table with machine ventilation. Continuous anesthesia was maintained by inhalation of Isoflurane and continuous intravenous infusion of Fentanyl. For relaxation intravenous bolus of Pipecuronium Bromide was applied. The animal was given Amoxycilline(875mg/125mg) with an enzyme inhibitor at the beginning of every procedure. During the surgical procedure we continuously monitored; arterial pressure, ECG, and saturation (tongue/lip). In the final step, the animal was euthanized in full anesthesia using bolus Thiopental (1-2g) + KCl 7,45% solution (40–50ml) i.v.The surgery took place in a specialized surgical theatre. After sedation using Intramuscular injection of All drug dosages were calculated based on actual animal weight.Stage IThe first group  was utilized to verify and optimize the technical aspects and functionality of the prototype sensor. In Stage I, we used hardware measuring at 7 wavelengths .The animal kidney was approached extraperitoneally using midline laparotomy, and the vessel supply of kidney was prepared for clamping. The prototype sensor was placed on the ventral plane of the kidney parenchyma. The sensor almost reached from one pole of the kidney to the other. This way, we could monitor the graft in its entire length.The kidney was then put through a series of cycles, during which kidney blood flow  was obstructed  for 10 minutes and later returned to normal. During testing, the quantities were recorded and evaluated. The second group  was utilized to establish and optimize sensor placement in the closed wound and to verify its function reliability over an extended period of time (48 hours).The animal kidney was approached extraperitoneally using midline laparotomy. The prototype sensor was placed on the exposed kidney capsule, located on the ventral plane of the organ. The system was fixed to the animal and the wound was closed. The animal was then allowed to move freely for 48 hours. During testing, the quantities were recorded and evaluated.Data taken from the first and the second group were used to optimize the measurement hardware setup, primarily to select a suitable subset of wavelengths. To simulate data that could be measured during artery/vein occlusion on a freely-moving animal, we added signals taken from both the first group  was utilized to simulate transplantation perioperative and early postoperative conditions and to verify system functionality and efficiency.We utilized surgical experience, gained from the first stage of the experiment, in the preparation of a unique laboratory porcine model of kidney transplantation. We performed an ipsilateral kidney autotransplantation to the iliac fossa using the retroperitoneal approach—Mimicking the conditions of human transplantation. The transplantation was completed with a standard full-thickness ureteroneocystostomy. The prototype sensor strap was then applied to the ventral plane of the graft. The connecting cord was passed through the abdominal wall (as would be done with drainage), and the battery and transmitter were fixed to the back of the animal. We fixed a second sensory system to the contralateral kidney in order to compare the performance of the transplanted graft and native kidney, thus eliminating any system perfusion complications.We then monitored the perfusion and effects of the wound oppression during closure (even simulating over-oppression).Two types of measurements were performed on each of the 20 laboratory animals.The first type was a 48 hour measurement after transplantation. During this period, the animal was in an awakened state and moving freely.The second type was a short-term (10+10 minute) measurement. The animal was put under anaesthesia and underwent arterial or venous clamping of the graft, which lasted 10 minutes. Subsequent changes in the measured wavelengths were recorded and analyzed and the blood flow was restored for another 10 minutes. These cycles were repeated to obtain more data.Prior to the animal being euthanized, we removed the sensor in order to verify that such action would not cause any complications, such as bleeding and tissue damage.All the data were analyzed, and a final prototype was prepared and completed with its own analytics software and chassis.The final kidney graft monitoring system Figs  and 4 coμA], at 650nm, 740nm, 850nm and 940nm, respectively. Every measuring point thus provides four signals, each corresponding to a single wavelength. The group of signals provided by a single measurement point is referred to as a channel. The arrangement with four measuring points allows better coverage of kidney graft volume and allows exclusion of unsuitable channels.The measurement probe contains four measuring points, each equipped with light sources of four wavelengths  and a photodiode that captures transmitted/reflected light. Specifically, the light emitters are HSMH-C170, MTSM0074-843-IR, VSMY1850 and APT1608F3C . The photodiode is TEMD5010X01. The measurement at different wavelengths is performed using a time division multiplex with sampling frequency at each wavelength equal to 25Hz. During each sampling period, the individual LEDs are sequentially activated, and the intensity of reflected/transmitted light is measured at each wavelength separately. The RMS/maximal currents through the individual diodes were 308/1500, 154/750, 308/1500, 1230/6000 [The measurement electronics generates signals for the light sources and captures weak signals from the photodiodes. It amplifies and digitizes them, and transfers the data to the central computer unit via a USB connection. The measurement probe sensor and the measurement electronics are encapsulated in epoxy and silicone (hardness Shore A 33).The computer unit houses a single board computer with a touch screen. It provides USB and Wi-Fi connectivity for remote control and data access. The single-board computer runs a Linux operating system and custom-developed software that acquires data from the measurement electronics and classifies the state of the monitored kidney. The results are presented on the touch screen via a graphical user interface that visualizes the spectral properties and photoplethysmographic waveforms of the organ Figs –8.rd order Chebyshev filters.The measured signals were divided into two groups: slow (<0.1Hz) changes that reflect the overall spectral properties of the transplanted kidney and faster (>0.3Hz) changes that show pulsatile signals related to the variations of blood oxygenation during a heart cycle. The low pass and high pass filters were implemented as 3The fast, high frequency signals with pulsatile changes can be interpreted as photoplethysmographic signals, and they are known to provide simple means of monitoring blood oxygen saturation. However, it was evident to us that these signals are highly susceptible to motion artifacts . These artifacts affect the optical coupling between the probe and the monitored tissue, and can affect the intensities of the measured transmitted/reflected light. This problem is more pronounced in our application where the probe is not firmly attached to the monitored tissue . The motion artifacts can either render the pulsatile waveform undistinguishable , they can cause mild baseline wonder , or they can affect the amplitudes of the pulsatile waveforms . Consequently, we determined that these signals are not sufficiently reliable to provide robust monitoring of blood perfusion. Even though these signals are monitored and visualized by our system, their primary use is limited to the initial setup of the measurement. Shortly after the kidney graft transplant  the presence of the pulsatile waveform can indicate which channels can be used for further monitoring; if no pulsatile changes are observed in a given channel, it is excluded from further monitoring.The slower, low frequency signals reflect the optical properties of the kidney graft on a time scale greater than the duration of a heartbeat. We found this to be a much more reliable indicator of kidney graft blood perfusion. We observed that the changes in these signals, caused by the decrease in the blood perfusion, is about one order of magnitude greater than the changes observable in higher frequency pulsatile waveforms. Consequently, the monitoring of slow changes is much less susceptible to the motion artifacts. We found that mechanical movement can still cause simultaneous increases and decreases in intensities in all channels; however, this effect is small enough to be effectively suppressed by normalizing the measurements with respect to the intensity measured at 940nm. Consequently, we based further classification on the slow changes in intensities measured at 650nm, 740nm and 850nm, normalized relatively to the intensity at 940nm . The normalization was performed by dividing the measured intensities at 650nm, 740nm and 850nm by the intensity at 940nm.Before the data were fed to the classifier, we computed their natural logarithm. Thus, the classified feature vector consisted of three numbers that were natural logarithms of normalized intensity at three wavelengths.The block diagram of the signal pre-processing path is shown in After initial processing, the data were subjected to the classification of detecting diminished blood perfusion. The following subsections describe the main elements of the classifier.Training data. To train the classifier, we used data from both the long-term and short-term measurements. These data were sufficient, so to avoid unnecessary overtraining, the long term measurements, during which the transplanted kidney failed, were not used in training.Testing data. All of the above-mentioned data were used in testing. To avoid overlearning, the leave-one-out cross-validation was used: to evaluate the performance on a chosen measurement, data obtained during this measurement were excluded from a training set. The classifier was retrained using the remaining data, and only then was it applied to the previously excluded measurement, evaluating its success rate.Classifier construction. To classify blood perfusion, we used a Bayesian classifier [assifier . The proThe classifier was constructed to identify 3 classes:correct blood perfusiondiminished blood perfusion due to a vein obstructiondecreased blood perfusion due to an artery obstructionTo avoid false-positive detections of diminished blood perfusion due to sensor movement or other short-term interferences, the classifier initiates a warning only if diminished blood perfusion is classified continuously for more than 3 minutes.We designed and constructed a new NIRS-based measurement system for kidney graft perfusion monitoring .We managed to obtain a total of 14 long-term measurement data sets . In otheFor the short-term data, we obtained 18 artery obstruction and 16 vein obstruction data sets .The measurement system manifested reliable performance in detecting diminished blood perfusion of transplanted kidneys.For long-term measurements, where the transplanted kidney performed correctly, the classifier showed a low false-positive rate . In caseFor long-term measurements, where transplanted kidney perfusion became obstructed, the classifier correctly identified all failures.For short-term measurements, intensity changes induced by vessel clamping were classified correctly . There wThe system proved to be safe for the laboratory animal. We did not encounter any complications with our experimental animals.The significance of the resulting classifier metrics can be verified by statistical testing. The individual metrics can be treated as statistics, and their respective p-values can be computed. We performed this computation using permutation tests , 19. TheHaving a means for an acute diagnosis of early vascular complications in the post-transplant period could improve overall results of kidney transplantation.Two approaches are recommended for the continuous monitoring of kidney graft perfusion: measuring of mechanical waves through Doppler ultrasound (DUS) and monitoring electromagnetic waves with near-infrared spectroscopy (NIRS).Blood flow can be continually measured using DUS with an invasive Doppler flow probe (DFP). This is currently an accepted method that is growing more common every year, even in transplantation medicine , 10. ThiExperimentally, NIRS has been used to monitor organ perfusion (even non-parenchymatous)  and evenWhen compared to a DFP, NIRS monitoring could prove to be a safer, cheaper and more specific solution.We believe our system would be of benefit, especially in situations with high rates of vascular complications .In the current stage presented study limitations are: an animal laboratory study results may vary a little, when implemented into human medicine. This being our initial study, we decided to use smaller group of animals to verify our systems functionality. When aiming for more significant statistical results, we will need to expand the cohort. Technically, our materials will need to be approved for human testing and long term in-vivo safety.Under laboratory conditions, our NIRS monitoring system proved reliable, with a very high degree of sensitivity as well as favorable specificity between arterial and venous causes.The system can be a useful tool for providing an early diagnosis of diminished blood perfusion of kidney transplants during the early postoperative period. However, further testing is still needed. We believe the application of our method in current human transplantation medicine is feasible, and our prototype is prepared for human testing.A similar system might also be of great use in various fields of surgery .S1 File(DOCX)Click here for additional data file.


P < 0.001), and more atherosclerotic plaques at the study onset (13 (26%) vs. 0 (0%), P < 0.001) compared to stage 2 CKD. A statistically significant 4-year increase in GFR  with non-significant CIMT alterations has been observed in stage 2 CKD. Furthermore, linear mixed effects analysis revealed significant decrease in GFR  with increase in CIMT  in stage 4 CKD. GFR and CIMT showed significant negative correlation in both CKD groups during all follow-up phases (P < 0.001). Furthermore, multiple linear regression analysis revealed significant independent prediction of CIMT by baseline GFR , while there was no significant prediction of CIMT with other covariates. In conclusion, this study demonstrates significant association of GFR and CIMT in non-diabetic stage 2 and stage 4 CKD during the 4-year follow-up.Patients with chronic kidney disease (CKD) have increased risk of cardiovascular events. However, the association of glomerular filtration rate (GFR) and carotid intima-media thickness (CIMT) in non-diabetic CKD patients is under-investigated. This prospective study was conducted at University Clinical Hospital Mostar over a 4-year period and enrolled a total of 100 patients with stage 2 and 4 CKD (50 patients per group). Stage 4 CKD group had significantly higher baseline CIMT values (1.13 ± 0.25 vs. 0.74 ± 0.03 mm,  Chronic kidney disease (CKD) is a progressive multifactorial disease characterized by permanent impairment of the renal function and/or structure . It reprCarotid intima-media thickness (CIMT), an ultrasound measure, has been shown to predict future cardiovascular events in the general population , while eThe study protocol was approved by the Ethics Committee of the University Clinical Hospital Mostar . All procedures were performed in accordance with the Helsinki declaration and its amendments. Written informed consent was obtained from all individual participants included in the study.This prospective longitudinal study was conducted at the Department of Nephrology of the University Clinical Hospital Mostar over a period from December 2014 to December 2018. All participants were informed about the procedures, course, aim of the study and follow-up protocol. Each subject was assessed upon study onset and further at each study timepoint (every year) for a total of four years. Baseline measurements involved detailed clinical, physical and laboratory evaluation, while the follow-up evaluation involved only estimate glomerular filtration rate (eGFR) and CIMT measurements .A total of 146 consecutive adult patients with stage 2 and 4 of CKD undergoing medical evaluation at the outpatient clinic of the Department of Nephrology were assessed for eligibility, while 100 patients were finally included in the research (50 patients per group). The diagnosis of CKD and the study groups were defined according to international guidelines, and classified into five stages with regards to the residual GFR: stage 1 (90–120 mL/min/1.73 m²), stage 2 (60–89 mL/min/1.73 m²), stage 3a (45–59 mL/min/1.73 m²), stage 3b (30–44 mL/min/1.73 m²), stage 4 (15–29 mL/min/1.73 m²) and stage 5 (<15 mL/min/1.73 m²). Estimated GFR (eGFR) was calculated using the Modification of Diet in Renal Disease (MDRD) equation . Exclusi2). Blood pressure was measured using a standard mercury sphygmomanometer, with a suitable arm, after the subject rested in a supine position for 15 min. Three measurements were performed with an interval of 10 minutes and the average measurements were taken as the final value of blood pressure.Detailed medical history was obtained from the anamnestic data and electronic health records, and all subjects underwent a detailed physical examination including anthropometric data assessment. Anthropometric measurements were conducted according to the conventional medical standards. A calibrated medical scale with an altitude meter  was used to measure body mass and height. BMI was calculated by dividing the value of body mass (kg) by the squared value of height  was determined by flow cytometry. Serum calcium and phosphorus were measured by a photometric staining test, while 25-hydroxyvitamin D (25(OH)D3) and parathyroid hormone (PTH) were determined with a microparticles chemiluminescence method (CMIA). Plasma C-reactive protein (CRP) levels were determined using a standard immunoturbidimetric method and represent standard CRP values. High density lipoprotein (HDL), low density lipoprotein (LDL) and triglycerides were determined using an enzyme staining test. Other biochemical analyses were measured by standard laboratory methods. Calcium levels were corrected according to albumin concentration, by increasing or decreasing total calcium levels for 0.8 units (mg/dL) with a decrease or increase of albumin levels for 1 unit (g/dL), respectively. The following formula was used: total calcium (mg/dL) + 0.80 × change in albumin from normal (g/dL) = corrected calcium (mg/dL).The degree of atherosclerosis was assessed using an ultrasound of the common carotid arteries. A type B linear probe with a frequency range of 5–10 MHz was utilized . Subjects were in a supine position and the carotid arteries were examined bilaterally in an oblique position from the front and rear. Three measurements were taken  by the same physician experienced in ultrasound imaging. The final CIMT value was calculated using an arithmetic mean of the bilateral measurements. Carotid plaque was defined as CIMT > 1.5 mm within the lumen of an artery which is in accordance with the European Mannheim consensus definition .P value. The statistical significance was defined as P < 0.05.Statistical software IBM SPSS Statistics  for Windows was used for statistical data analysis. Data were expressed as mean ± standard deviation or median (interquartile range) for continuous variables, and as whole numbers and percentage for categorical variables. The Kolmogorov–Smirnov test was used for normality of data distribution. Student’s t-test was used for the analysis of parametric continuous data, and Mann-Whitney U test was used for the analysis of non-parametric continuous data. Chi-square and Fisher’s test were used for analysis of categoric variables. Pearson’s correlation served for assessment of correlation between selected variables. Multivariable linear regression analysis with enter selection algorithm was used to determine the relative importance of independent variables  in prediction of CIMT levels. The linear mixed effect model with the unstructured covariance structure and restricted maximum likelihood algorithm were used for the within-subjects comparison of the eGFR and CIMT over the follow-up. Results of the linear mixed effects model analysis were expressed as parameter estimates with 95% confidence interval (CI), t-value, Bayesian information criteria and the corresponding P < 0.001; 149.30 ± 18.24 vs. 127.80 ± 9.80 mmHg, P < 0.001; and 27.46 ± 3.36 vs. 22.56 ± 2.67 kg/m2, P < 0.001, respectively). Other baseline characteristics are shown in There was no significant difference in demographic and anthropometric characteristics between studied groups, except in age, systolic blood pressure and BMI which were higher in stage 4 CKD group  vs. 48  years, 2) and gradual decrease in stage 4 CKD group (26.68 ± 2.95 vs. 25.10 ± 3.20 vs. 23.84 ± 3.33 vs. 22.57 ± 2.29 vs. 21.23 ± 1.06 mL/min/1.73 m2)   , which p< 0.001) .P < 0.001), and stage 4 CKD group had statistically significantly more atherosclerotic plaques at the study onset (13 (26%) vs. 0 (0%), P < 0.001) (P < 0.001)  . During < 0.001)  which pr< 0.001) . There w< 0.001) . There was statistically significant negative correlation of eGFR and CIMT at the beginning of the study and during each time point of the follow-up in both stage 2 and stage 4 CKD group .r = −0.77, P < 0.001), BMI , CRP , PTH  and phosphorus . On the contrary, baseline eGFR exhibited statistically significant positive correlation with cholesterol, calcium and vitamin D levels , cholesterol , CRP , PTH  and phosphorus . On the other hand, baseline CIMT exhibited statistically significant negative correlation with calcium and vitamin D levels  in the total cohort, while there was no significant prediction of CIMT with other variables  and more newly developed atherosclerotic plaques (15 (30%) vs. 0 (0%), P < 0.001), while there was no statistically significant difference in the rates of progression to the next CKD stage or hemodialysis. Specifically, one patient died in the third year and four patients died in the fourth year of the study with the death cause being acute myocardial infarction and stroke. Finally, stage 4 CKD group had significantly higher occurrence of the composite outcome (15 (30%) vs. 6 (12%), P = 0.027) compared to stage 2 CKD  vs. 0 (0%), ge 2 CKD .The current longitudinal study demonstrates a significant association of eGFR and CIMT in non-diabetic patients with stage 2 and stage 4 CKD during the 4-year follow-up. Previous studies have investigated CIMT in relation to renal function in diabetic CKD population ,24, dialThis study has several major findings. First, non-diabetic patients with stage 4 CKD exhibited significant progressive deterioration of the residual renal function, while the stage 2 CKD group showed dominantly stable function over the 4-year follow-up. Second, non-diabetic stage 4 CKD patients displayed significant gradual expansion of the atherosclerotic burden during the 4-year follow-up as measured by CIMT, whereas stage 2 CKD patients showed minimal non-significant time-dependent CIMT alterations. Furthermore, there was a statistically significant negative correlation of eGFR and CIMT, independently of the study period. Finally, stage 4 CKD patients are significantly more prone to all-cause mortality and the development of new atherosclerotic plaques.Previous studies have shown differing findings about the association of CIMT and eGFR. Specifically, Chhajed et al. found no statistically significant correlation between CIMT and eGFR in CKD patients , while ASeveral other studies have investigated GFR and CIMT in a solely diabetic CKD population. CIMT has shown a strong and independent prediction of cardiovascular mortality in this population, while exhibiting significant negative correlation with eGFR ,24. HoweThe importance of CIMT measurement across different spectrums of CKD has not been fully established. Buscemi et al. have demonstrated a significant negative correlation of GFR and CIMT in patients with near normal or mildly decreased renal function , but theAdditionally, stage 4 CKD patients had a significant incidence rate of atherosclerotic plaques during the study period, which are predictive of future cardiovascular events . This fiThis study has several limitations. A healthy control group could serve as the direct longitudinal comparison with CKD patients, especially with stage 2 CKD, and its absence represents a limitation. Furthermore, some potentially confounding clinical and laboratory parameters were not assessed during the follow-up which prevents longitudinal adjustment analyses, and possible confounding effects could not be eliminated. Moreover, we did not address the different causes of chronic renal failure and the results of our study are not applicable to patients with CKD stages other than 2 and 4. Despite the longitudinal study design, the real causality cannot be established, but rather the association of GFR and CIMT. Finally, this study does not provide insights into the long-term association of GFR and CIMT (>4 years) which should be assessed in different study designs.In conclusion, this study demonstrates a significant association of GFR and CIMT in non-diabetic stage 2 and stage 4 CKD patients during the 4-year follow-up. Patients with stage 4 CKD have progressive renal deterioration, higher all-cause mortality and develop new atherosclerotic plaques, in comparison to stage 2 CKD group. Future studies are necessary to determine causality between renal function and atherosclerotic process in these patient groups.