O exame físico permite a avaliação prognóstica de pacientes com insuficiência cardíaca (IC) descompensada, porém não é suficientemente confiável e depende da experiência clínica do profissional. Considerando as respostas hemodinâmicas a situações do tipo “luta ou fuga” tais como a admissão no serviço de emergência, foi proposto o índice hemodinâmico agudo (IHA), calculado a partir da frequência cardíaca e pressão de pulso. avaliar a capacidade prognóstica intra-hospitalar do IHA na IC descompensada.Receiver Operating Characteristic), de estatística c e de regressão multivariada, assim como o critério de informação de Akaike, para testar a capacidade prognóstica do IHA. O valor-p < 0,05 foi considerado estatisticamente significativo. estudo prospectivo, multicêntrico e observacional baseado no registro BREATHE, incluindo dados de hospitais públicos e privados no Brasil. Foram utilizadas análises ROC . Um baixo IHA (≤ 4 mmHg⋅bpm) foi encontrado em 80% dos pacientes falecidos. O risco de mortalidade intra-hospitalar em pacientes com baixo IHA foi 2,5 vezes maior que aquele para pacientes com IHA > 4 mmHg⋅bpm. O IHA foi capaz de predizer independentemente a mortalidade intra-hospitalar na IC aguda descompensada  mesmo depois dos ajustes para comorbidades e uso de medicamentos . O IHA é capaz de predizer independentemente a mortalidade intra-hospitalar na IC aguda descompensada. Esse índice simples e realizado à beira do leito pode se mostrar útil em serviços de emergência. :77-86) Na realidade, a literatura apresenta resultados controversos, indicando que uma frequência cardíaca alta no momento da admissão pode estar relacionada a um prognóstico melhor ou pior.22 Nesse contexto, aumentos na pressão de pulso e frequência cardíaca são esperados, aumentando a perfusão nos músculos esqueléticos e órgãos vitais.A admissão aguda no serviço de emergência é uma situação de estresse onde se esperam respostas autonômicas que preparam o corpo para uma situação de luta ou fuga.Com base nas respostas hemodinâmicas fisiológicas inerentes a situações de luta ou fuga, propõe-se o cálculo do índice hemodinâmico agudo (IHA) a partir da frequência cardíaca e pressão de pulso. Nossa principal hipótese é que o IHA poderia representar um parâmetro prognóstico objetivo e intra-hospitalar a ser usado na descompensação aguda de pacientes com IC com fração de ejeção reduzida (ICFER). Portanto, o objetivo deste estudo foi avaliar a capacidade prognóstica intra-hospitalar do IHA na ICFER aguda descompensada.24 um estudo observacional transversal tipo registro da IC aguda com seguimento longitudinal que ocorreu de fevereiro de 2011 a dezembro de 2012. Para inclusão no registro, pacientes deveriam possuir mais de 18 anos e ter sido admitidos com IC descompensada; os pacientes não poderiam ter sido submetidos a cirurgia de revascularização miocárdica ou intervenção coronária percutânea no mês anterior, nem ter sido admitidos com um diagnóstico de sepse. Os critérios de Boston foram utilizados para a confirmação da IC.25 A participação no registro não requeria nenhum regime de tratamento especial. Os métodos detalhados, assim como os critérios de inclusão e exclusão, estão disponíveis na literatura.24 As informações sobre cada paciente foram incluídas num formulário de registro individual, disponívelonline.Esta análise foi baseada no I Registro Brasileiro de Insuficiência Cardíaca (BREATHE),Este estudo incluiu a análise da admissão hospitalar e do seguimento dos pacientes até a alta médica, morte ou transferência para outro hospital (o que foi verificado primeiro) dos pacientes com ICFER aguda descompensada. O desfecho primário do estudo foi a mortalidade intra-hospitalar.Todos os pacientes do registro com evidência de fração de ejeção do ventrículo esquerdo menor que 40% foram incluídos nesta análise, exceto aqueles com informações faltantes . Indivíduos com ritmo cardíaco controlado por marca-passo também foram excluídos, visto que não era esperado que suas frequências cardíacas possuíssem um controle autonômico .Frequência cardíaca e pressão arterial sistólica e diastólica na admissão foram obtidas a partir da base de dados do registro e utilizadas no cálculo das variáveis derivadas da seguinte maneira: pressão de pulso = pressão arterial sistólica – pressão arterial diastólica; pressão de pulso proporcional = pressão de pulso / pressão arterial sistólica; IHA= (Pressão de pulso x frequência cardíaca) / 1000Este estudo está de acordo com os princípios da Declaração de Helsinque. O estudo foi aprovado pelo Hospital do Coração, São Paulo (registro 144/2011) e pelo comitê de ética em pesquisa de cada instituição participante. Todos os pacientes assinaram um termo de consentimento esclarecido antes do recrutamento.Inicialmente, utilizado o teste de Shapiro Wilk para verificar a normalidade da distribuição dos dados, validando o uso da estatística paramétrica. Variáveis contínuas foram descritas como médias e desvios-padrão, e variáveis categóricas foram descritas como proporções. Dados clínicos e demográficos dos pacientes que vieram a óbito durante a hospitalização  e daqueles que receberam alta médica (vivos) foram comparados por um teste t de Student não-pareado ou qui-quadrado. Um valor-p bicaudal < 0,05 foi considerado estatisticamente significativo.Receiver Operating Characteristic) usando a mortalidade intra-hospitalar como desfecho principal. O valor de corte do IHA foi definido pelo 50º percentil de sua distribuição. Sensibilidade, especificidade, e área sob a curva ROC (AUC) foram estabelecidas para cada valor de corte. Estatística c foi usada para comparar a capacidade prognóstica dos valores de corte de frequência cardíaca e pressão arterial com os valores de corte de IHA.Depois de verificar a distribuição normal, os percentis 25º, 50º e 75º de frequência cardíaca e pressão arterial sistólica e diastólica foram utilizados na construção de curvas ROC software STATA versão 14.2  foi utilizado em todas as análises estatísticas e gráficos.OO registro BREATHE incluiu 463 pacientes com ICFER admitidos em serviços de emergência no Brasil , com umOs pacientes falecidos apresentavam mais comorbidades, assim como frequência cardíaca e pressão arterial sistólica e diastólica mais altas que aqueles que sobreviveram. Considerando o 50º percentil do IHA como valor de corte (4 mmHg⋅bpm), quase 80% dos pacientes falecidos possuíam um baixo IHA.Visto que o cálculo do IHA inclui valores de frequência cardíaca e pressão arterial, foram comparadas as AUC de IHA ≤ 4 mmHg⋅bpm e de diferentes valores de corte de frequência cardíaca e pressão arterial sistólica e diastólica . IHA ≤ 27 Diferentes razões contribuem para uma alta mortalidade a curto prazo de pacientes com IC aguda descompensada, como idade, comorbidades e tempo entre início dos sintomas e admissão hospitalar.27 Visto que o manejo de pacientes com IC aguda pode incluir procedimentos invasivos e de alto custo, como o suporte circulatório mecânico, a validação de índices prognósticos que possam auxiliar em decisões terapêuticas é de extrema importância.28Este estudo apresentou o IHA e demonstrou que este é um preditor independente de mortalidade intra-hospitalar em pacientes com ICFER aguda descompensada. A mortalidade intra-hospitalar de pacientes com IC aguda descompensada é elevada, como demonstrado pelo registro BREATHE e por estudos realizados em outros países.2 O treinamento destes profissionais e a disponibilidade de recursos nestes ambientes pode variar substancialmente. Quando somados à diversidade de casos de pacientes, estes aspectos tornam difícil a criação de escores prognósticos que possam ser utilizados amplamente. Por exemplo, apesar do recente interesse no uso de biomarcadores,29 a avaliação destes pode não estar disponível em estabelecimentos de saúde afastados dos grandes centros ou com poucos recursos. Nohria et al.6 apresentaram uma abordagem clínica prática para classificar os pacientes em perfis hemodinâmicos, permitindo a predição do prognóstico e guiando o tratamento da IC aguda. Esta abordagem depende da experiência do médico responsável9 e pode perder sua utilidade quando se trata de um médico não-especialista em IC. Nossos resultados corroboram a falta de acurácia do exame físico cardiovascular,9 visto que 11% dos pacientes foram classificados como de perfil hemodinâmico A e ainda assim considerados casos de IC aguda descompensada.A IC aguda descompensada pode ser manejada por especialistas em IC, cardiologistas, intensivistas, médicos emergencistas ou clínicos; em serviços de emergência, enfermarias ou unidades de tratamento intensivo.12 Estudos anteriores tentaram utilizar estes parâmetros como fatores prognósticos na IC aguda descompensada. A relação entre frequência cardíaca e prognóstico é conhecida há décadas, e com o advento dos betabloqueadores e, mais recentemente, da ivabradina, frequências cardíacas baixas são um alvo no tratamento da IC estável.19 Além disso, a incompetência cronotrópica também é um marcador de risco: pacientes cujas frequências cardíacas não aumentam durante o exercício físico têm prognósticos piores que aqueles com frequências cardíacas de reserva normais, mesmo com o uso de betabloqueadores.21 Apesar de estudos terem determinado o aumento esperado na frequência cardíaca durante um teste de esforço,21 não há valores de referência para o aumento da frequência cardíaca em situações do tipo “luta ou fuga” como é o caso das admissões em serviços de emergência. Pacientes japoneses com IC aguda descompensada admitidos com frequências cardíacas acima de 120 bpm apresentaram menores índices de mortalidade que aqueles admitidos com frequências cardíacas menores.15 Entretanto, uma alta frequência cardíaca foi considerada um preditor independente de mortalidade a curto prazo em pacientes com IC aguda descompensada em outros estudos.31A medição da frequência cardíaca e a pressão arterial está disponível em praticamente qualquer estabelecimento de saúde, tem boa acurácia e requer treinamento mínimo.14 observou que pressão arterial sistólica abaixo de 120 mmHg identificava pacientes com IC aguda descompensada que possuíam um prognóstico ruim apesar dos tratamentos. Baixa pressão arterial sistólica também indicou risco a curto prazo numa coorte europeia.13 Neste estudo, a pressão arterial abaixo de 120 mmHg não estava independentemente relacionada à mortalidade de acordo com a análise multivariada. Pacientes do registro BREATHE eram mais jovens, e os protocolos de tratamento estavam mais atualizados que aqueles utilizados nos outros estudos (quase uma década antes). Além disso, esses dois estudos14 incluíram pacientes com fração de ejeção preservada ou reduzida, e o valor prognóstico da pressão arterial pode variar de acordo com a fração de ejeção do ventrículo esquerdo.32 Baixa pressão de pulso foi definida como um preditor independente de mortalidade na IC aguda descompensada pelo estudo VMAC-HF.33 O tratamento da IC evoluiu substancialmente desde então, o que pode explicar a falta de poder prognóstico da pressão de pulso em nosso estudo.O registro OPTIMIZE-HFA interação intrínseca entre pressão arterial e frequência cardíaca e como elas são influenciadas por medicamentos para IC pode ter influenciado os resultados de estudos prévios considerando cada um destes parâmetros. Até onde sabemos, este é o primeiro estudo a apresentar um índice que combina a análise da frequência cardíaca e pressão de pulso de pacientes com IC aguda descompensada e demonstrar que sua capacidade como ferramenta prognóstica é superior àquelas da frequência cardíaca ou pressão arterial sozinhas.Esta análise possui limitações. Primeiramente, a mortalidade intra-hospitalar foi baseada no registro feito por pesquisadores e não foi confirmada. Na realidade, registros são estudos observacionais, e análises quanto à adequação do tratamento a cada paciente não estavam no escopo de nosso estudo. Considerando que nosso principal objetivo foi analisar a utilidade de um índice facilmente obtido no momento da admissão do paciente no setor de emergência e que os testes de troponina e BNP  não estão disponíveis em todos os estabelecimentos de saúde brasileiros, parâmetros laboratoriais não foram incluídos em nosso modelo.12 Além disso, o fato de não existir uma padronização neste caso aumenta a aplicabilidade clínica do nosso índice, já que reflete resultados reais.Os dados do registro não foram obtidos através de um protocolo padronizado, portanto as medições de pressão arterial e frequência cardíaca podem ter sido realizadas com equipamentos diferentes. De qualquer maneira, estes são sinais vitais que requerem treinamento mínimo para sua medição.19 que poderiam influenciar o IHA.É importante notar que os resultados apresentados neste estudo se restringem a pacientes com fração de ejeção reduzida. O estudo foi conduzido entre 2011 e 2012, antes da aprovação de medicamentos novos como a ivabradina e sacubitril-valsartana,23 Embora a generalização de nossos resultados possa ser limitada, é importante enfatizar que os dados clínicos e demográficos dos pacientes incluídos neste estudo são semelhantes aos de outras coortes.31A população brasileira é muito diversa quanto à etnia e acesso a estabelecimentos de saúde, e este estudo incluiu hospitais públicos e privados de todas as regiões do país.É importante ressaltar que a AUC da análise do IHA foi relativamente pequena. Mesmo assim, sua sensibilidade foi boa e pode ser útil na orientação de médicos emergencistas na triagem de pacientes.Diferentes fatores prognósticos têm sido propostos para a IC aguda descompensada. Entretanto, muitos dependem de biomarcadores, treinamento de pessoal e recursos tecnológicos, que nem sempre estão disponíveis. O IHA consiste num fator prognóstico de mortalidade intra-hospitalar prático, objetivo e facilmente obtido em pacientes com IC aguda descompensada. Futuros estudos prospectivos deverão avaliar a reprodutibilidade destes resultados em outras populações. 1Although previous studies have shown that treatment by a HF specialist can lead to better results, most cases of acute decompensated HF are originally evaluated and managed by emergency physicians3in facilities with different levels of resource availability.Heart failure (HF) is one of the main reasons for emergency admissions in the Western world.5Physicians evaluate congestion and perfusion from the patient’s history and a physical examination, assigning hemodynamic profiles that guide therapy and provide prognostic information in an acute HF setting.6Although practical, the physician’s assessment of perfusion lacks reliability7and depends on clinician experience,9providing subjective information.10Therefore, objective prognostic parameters that can be easily obtained in the emergency room would be useful in the management of acute HF.Despite recent advances in technology and medical devices, the physical examination remains the cornerstone of the evaluation of patients with HF.12Systolic blood pressure is an independent predictor of in-hospital and post-discharge outcomes in acute heart failure.14Additionally, low blood pressure and narrow proportional pulse pressure are markers of low perfusion.9Blood pressure and heart rate are parameters that can be easily obtained by any healthcare professional with good reproducibility and accuracy.17Although low resting heart rates reduce risk in patients with stable chronic HF with reduced ejection fraction (HFrEF),19the ability to increase heart rate during a “fight or flight” reaction certainly confers good prognosis,21regardless of the use of beta-blockers.The relationship between admission resting heart rate and the prognosis of patients with HF is not as straightforward. In fact, the literature is controversial, showing that a high admission heart rate can be related to worse or better prognoses.22Increases in pulse pressure and heart rate are thus expected in this scenario, augmenting perfusion in skeletal muscles and vital organs.Acute admission to the emergency room is a stressful situation, expected to elicit autonomic responses that prepare the body to fight or flight.Based on the physiological hemodynamic responses inherent to “fight or flight” situations, we have proposed the calculation of the acute hemodynamic index (AHI) using heart rate and pulse pressure. Our main hypothesis was that AHI could be an objective in-hospital prognostic parameter to be used in patients with acute decompensated HFrEF. Therefore, we aimed to evaluate the in-hospital prognostic ability of AHI in acute decompensated HFrEF.24a cross-sectional, observational acute HF registry with longitudinal follow-up that happened from February 2011 to December 2012. For inclusion in the registry, patients should be over 18 years old and have been admitted with decompensated HF; patients should not have been submitted to a coronary artery bypass graft of percutaneous coronary intervention in the previous month or have been admitted with a sepsis diagnosis. Boston criteria were used for HF confirmation.25Participation in the registry did not require any special treatment regimen. Detailed methods, as well as exclusion and inclusion criteria, have been previously described.24Data on each patient are available online in individual registration forms.This analysis is based on the I Brazilian registry of HF (BREATHE Registry),This study includes the analysis of patients with acute decompensated HFrEF from hospital admission and follow-up until discharge, death, or transfer to another hospital (whichever happened first). The primary endpoint of the study was in-hospital mortality.All patients in the registry with evidence of left ventricle ejection fraction < 40% were included in the present analysis, except those with missing information . Individuals with pacemaker-controlled heart rhythm were also excluded, as their heart rate was not expected to be autonomic-driven .Heart rate and systolic and diastolic blood pressure at admission were available from the registry database and were used for calculating derived variables as follows: pulse pressure = systolic blood presssure – diastolic blood pressure; proportional pulse pressure = pulse pressure / systolic blood pressure; AHI= (pulse pressure x heart rate) / 1000.This investigation conforms to the principles outlined in the Declaration of Helsinki. The study was approved by the Hospital do Coração, São Paulo (registry 144/2011) and the Institutional Review Board of each participating institution. All patients signed an informed consent form before enrollment.Initially, a Shapiro-Wilk test was used to verify the normality of data distribution and validate the use of parametric statistics. Continuous variables were reported as means and standard deviations, while categorical variables were reported as proportions. Clinical and demographic data from patients who died during the hospitalization period (deceased) and those who were successfully discharged  were compared using unpaired Student’s t-tests or chi-squared tests. A two-sided p-value < 0.05 was considered significant.After verifying a normal distribution, the 25th, 50th, and 75th percentiles of heart rate and systolic and diastolic blood pressure were used to construct receiver-operating characteristic (ROC) curves using in-hospital mortality as the main outcome. The cut-off value defined for the AHI was its 50th percentile. Sensitivity, specificity, and area under the ROC curve (AUC) were reported for each cut-off value. C-statistics were used to compare the prognostic ability of heart rate and blood pressure cut-off values to the AHI cut-off values.Regression analyses were performed after verifying for linear relationships, multivariate normality, homoscedasticity, and the absence of multicollinearity and autocorrelation.26was used to compare multiple regression models. All statistical analyses and graphs were performed using STATA 14.2 .Multiple linear regression analyses were performed to test the independent prognostic ability of each significant cut-off value regarding heart rate, blood pressure, and AHI. This analysis included variables with statistical significance according to the previously cited unpaired Student’s t-tests or chi-squared tests. As laboratory results were not available for all patients, they were not included in the regression analysis. Akaike’s information criterion (AIC)The BREATHE registry included 463 patients with HFrEF admitted to emergency services in Brazil , with aDeceased patients presented more comorbidities and higher values of heart rate and systolic and diastolic blood pressure when compared to survivors. Considering the AHI’s 50th percentile, its cut-off value was 4 mmHg⋅bpm; almost 80% of the deceased patients had a low AHI.As the AHI calculation included heart rate and blood pressure values, we compared the AUC of AHI ≤ 4 mmHg⋅bpm as a cut-off value with the AUC of different cut-off values of heart rate and systolic and diastolic blood pressure . AHI ≤ 27Different reasons for this high short-term mortality include age, comorbidities, and the delay between symptom onset and hospital admission.27Since the management of patients with acute HF may include invasive and high-cost procedures such as circulatory support, it is critical to validate prognostic factors that can help guiding therapeutic decisions.28The present study introduced the AHI and demonstrated that it is an independent predictor of in-hospital mortality in patients with acute decompensated HFrEF. In-hospital mortality in patients with acute decompensated HF is high, as shown by this Brazilian registry and by studies conducted in other countries.2The physician’s experience and the available resources can vary substantially. Together with the patients’ diversity, these aspects hinder the production of widely applicable prognostic scores. Despite the recent attention received by biomarkers,29for example, their verification may not be available in remote or low-income health facilities. Nohria et al.6have introduced a practical clinical approach for categorizing patients with hemodynamic profiles, thus enabling prognosis prediction and guiding treatment in acute HF settings. This approach relies on clinician experience9and may be less useful when considering non-HF specialists. Our results corroborate the lack of accuracy of cardiovascular physical examinations,9since 11% of the patients were classified as hemodynamic profile A despite having acute decompensated HF.Acute decompensated HF can be managed by HF specialists, general cardiologists, intensivists, emergency physicians, or internists; this can be performed in emergency departments, hospital wards, or intensive care units.12Previous studies have tried to use blood pressure and heart rate as prognostic factors in acute decompensated HF; the relationship between heart rate and prognosis in heart disease has been known for decades. Since the emergence of therapies using beta-blockers and more recently, ivabradine, low heart rates have been considered a target in the treatment of stable HF.19On the other hand, chronotropic incompetence is also a risk marker. Patients whose heart rates do not increase during exercise have worst prognoses than those with normal heart rate reserves, even with the use of beta-blockers.21Although previous studies have determined the expected increase in heart rate during an exercise test,21no normality values have been established for heart rate increases during “fight or flight” situations such as the admission to emergency rooms. Japanese patients with acute decompensated HF admitted with heart rates above 120 bpm presented lower mortality indices than those with lower heart rates.15Conversely, high heart rate was considered an independent predictor of short-term mortality in patients with acute decompensated HF in other studies.31Heart rate and blood pressure measurements are available in virtually any healthcare facility with good accuracy and requiring minimal training.14registry found that systolic blood pressure values below 120 mmHg characterized patients with acute decompensated HF who had poor prognoses despite medical therapy. Low systolic blood pressure levels also indicated high short-term risk in a European cohort.13In our study, blood pressure below 120 mmHg was not independently related to mortality in a multivariate analysis. Patients in the BREATHE registry were younger, and treatment protocols were more updated when compared to those used in studies conducted almost a decade earlier. Furthermore, both studies14included patients with preserved and reduced ejection fraction, and the prognostic value of blood pressure is known to vary according to the left ventricular ejection fraction.32Low pulse pressure was defined as an independent predictor of mortality in acute decompensate HF by the VMAC-HF study.33Since the publication of this trial, HF therapy has evolved substantially, which may explain the lack of prognostic power of pulse pressure in our patients.The OPTIMIZE-HFThe intrinsic interaction between blood pressure and heart rates and how they are affected by HF medications may have influenced the results of previous investigations on each of these parameters. To our knowledge, this is the first study to introduce an index that analyzes both heart rate and pulse pressure in patients with acute decompensated HF; moreover, we have shown that the prognostic ability of the AHI is higher than that of heart rate or blood pressure alone.The present analysis has limitations. First, in-hospital mortality was based on investigator reports instead of being adjudicated. In fact, registries are observational studies and analyzing the treatment delivered to each patient was not within the scope of our study. As our main objective was to analyze the usefulness of an easily obtained index to be applied as soon as patients arrive in the emergency room, and considering the unavailability of troponin and brain natriuretic peptide (BNP) tests in some Brazilian health facilities, laboratory parameters were not included in the model.12Additionally, the fact that the registry had no standardized assessment methods enhances the clinical applicability of our study, as it shows realistic results.Data in the registry was not obtained by any specific protocol, and blood pressure and heart rate measurements may have been performed using different equipment. Nevertheless, blood pressure and heart rate are vital signs that require minimal training for their measurement.19which could influence AHI values.The present results are restricted to patients with HFrEF. The study was conducted from 2011 to 2012, before the approval of new HF medications as ivabradine and salcubitril-valsartan,23Although the generalization to other populations may be limited, we highlight that the demographical and clinical data of patients included in this registry are very similar to those of other cohorts.31The Brazilian population is very diverse regarding ethnicity and access to health care facilities. The study included private and public hospitals in all regions of the country.Finally, the AUC in the ROC analysis of the AHI was relatively low. Nevertheless, its sensitivity was quite good and this may be useful to guide emergency physicians while triaging patients.Different prognostic factors have been proposed in acute decompensated HF but rely on biomarker measurement, medical staff training, and technology; these may not be widely available. The AHI is a practical, objective, and easily obtained prognostic factor for in-hospital mortality in patients with acute decompensated HF. Further prospective studies should evaluate the reproducibility of these results in other populations.


IntroductionWe hypothesized that the nephroprotective and diuretic effects of carperitide are effective in patients with pulmonary hypertension. We examined the presence of preoperative pulmonary hypertension and the effects of carperitide.MethodsIn this retrospective cohort study, we included patients aged 20 years or older who received carperitide during cardiovascular surgery and were admitted to the postoperative intensive care unit. We used hospital data from March 2019 to September 2021. The outcomes were the incidence of acute kidney injury, the number of patients using renal replacement therapy in the intensive care unit, urine volume in the first 24 hours after surgery, and the difference in serum creatinine concentrations between before and after surgery. After adjusting for confounding factors by multivariate analysis, we compared the difference in outcomes with and without preoperative pulmonary hypertension (systolic pulmonary artery pressure ≥36 mmHg).ResultsThe study included 244 patients, with 72 (29.5%) in the pulmonary hypertension group and 172 (70.5%) in the control group. Acute kidney injury occurred in eight (11.1%) patients in the pulmonary hypertension group and in 18 (10.5%) patients in the control group, with no significant difference by logistic regression analysis . Additionally, the use of renal replacement therapy, urine volume at 24 hours postoperatively, and the difference in serum creatinine concentrations were not different between the two groups.ConclusionsOur results suggest that the effect of carperitide during cardiovascular surgery is not affected by the presence or absence of pulmonary hypertension. Acute kidney injury (AKI) remains a common complication after cardiovascular surgery ,2. The mCarperitide has multiple independent mechanisms of action, including vasodilation, inhibition of sodium reabsorption, reduction of renin activity, angiotensin II concentrations, blood aldosterone concentrations, and sympathetic inhibition . ContinuWe hypothesized that in patients with pulmonary hypertension before cardiovascular surgery, the vasodilatory and diuretic effects of carperitide are beneficial, and the nephroprotective effects of carperitide are more effective than in those without pulmonary hypertension. Therefore, this study aimed to investigate whether there is a difference in the nephroprotective effect of carperitide during cardiac surgery between patients with preoperative pulmonary hypertension and those without pulmonary hypertension using our database.We conducted a retrospective cohort study at Hamamatsu University Hospital  between March 2019 and September 2021. The study protocol was approved (21-238) by the Ethics Review Board of Hamamatsu University School of Medicine. The Ethics Review Board waived the requirement for written informed consent because of the retrospective design of the study and the absence of follow-up. This study was conducted according to the STROBE checklist and complied with the Declaration of Helsinki 1964 and its later amendments.We included adult patients aged 20 years or older who received carperitide  during cardiovascular surgery and were admitted to the intensive care unit (ICU) postoperatively. The exclusion criteria were as follows: (i) patients who did not have right ventricular catheterization or echocardiography before surgery; (ii) patients younger than 20 years of age; (iii) patients with emergency surgery; and (iv) patients who were not administered carperitide during surgery. According to the following procedure, all patients were classified into the pulmonary hypertension group and the control group without pulmonary hypertension. In this study, we used systolic pulmonary artery pressure as measured by preoperative right heart catheterization. In patients who did not undergo right heart catheterization, the estimated systolic pulmonary artery pressure was calculated from the tricuspid regurgitation pressure gradient and the estimated right atrial pressure on echocardiography . PatientDemographic data  were automatically extracted from our hospital data registered in the Japanese intensive care patient database (JIPAD) , which iThe primary outcome was the incidence of AKI based on the Kidney Disease: Improving Global Outcomes (KDIGO) criteria within 24 hours after surgery . SecondaStatistical analysesWe calculated the median and interquartile range for continuous data and the number and percentage for categorical data. The Mann-Whitney test was applied to compare the mean values of continuous variables. Categorical variables were analyzed using the χ2 test or Fisher’s exact test. For the primary outcome, the incidence of AKI was analyzed using a multivariate logistic regression model. Sensitivity analysis was also based on multivariate logistic regression. The multivariate linear regression model was used to evaluate the secondary outcomes of continuous variables (urine volume within 24 hours after surgery and the difference in serum creatinine concentrations between before and after surgery). Additionally, the secondary outcomes of binary variables (number of patients using RRT) were evaluated using Fisher’s exact test because of the small number of events. Two-sided p values of <0.05 were considered statistically significant. All statistical analyses were conducted using Stata/BE 17.0 .Six hundred seventy-nine patients were admitted to the ICU after cardiovascular surgery at Hamamatsu University Hospital during the study period. The exclusion criteria were applied (see Figure The characteristics and surgical data of the patients included in this study are shown in Table Figure The secondary outcomes are shown in Table We aimed to investigate whether there is a difference in the nephroprotective effect of carperitide during cardiac surgery between patients with preoperative pulmonary hypertension and those without pulmonary hypertension. The incidence of AKI, which was the primary endpoint, was not different between the two groups and was robust in a sensitivity analysis. The number of patients using RRT in the ICU, urine volume within the first 24 hours after surgery, and the difference in serum creatinine concentrations were not different between the two groups. Therefore, carperitide had a similar effect according to the presence or absence of preoperative pulmonary hypertension in this study.To the best of our knowledge, this study is the first to investigate whether there is a difference in the nephroprotective effect of carperitide according to the presence or absence of pulmonary hypertension. Although our hypothesis focusing on pulmonary hypertension was rejected, previous studies have reported preventing AKI by stratifying risk in the perioperative period of cardiac surgery ,21. CarpIn this study, there were no differences in the number of patients using RRT, urine volume within 24 hours after surgery, or the difference in serum creatinine concentrations before and after surgery between patients with and those without pulmonary hypertension. Previous reports have suggested the efficacy of carperitide regarding the number of RRTs performed, urine volume, and serum creatinine values 24 hours after surgery ,25-27. WThe definition of pulmonary hypertension has not been standardized. We set the cutoff for pulmonary hypertension at 36 mmHg and also used 50 mmHg in a sensitivity analysis on the basis of the guideline . HoweverThe present study has several limitations. The first limitation is that because this study was a single-center, retrospective study, there was no protocol for the method of carperitide administration. However, the carperitide dosage used at our institution was low, which is mostly consistent with previous reports -27. AddiWe focused on the vasodilatory effect of carperitide and hypothesized that carperitide is more effective in patients with pulmonary hypertension than in those without pulmonary hypertension. Unfortunately, stratification by the presence or absence of pulmonary hypertension did not result in any differences in renal function outcomes, including the incidence of AKI, which is a measure of the nephroprotective effect of carperitide. However, the risk stratification of developing AKI has been a subject of recent investigation, and further research is warranted.


Objective: Several epidemiological studies suggest that the preservation of the physiological circadian rhythm of blood pressure or its disruption affects the extent of the organ damage developed by the patient. If we classify the circadian rhythm of blood pressure into four nocturnal profiles, significant differences emerge in terms of organ damage burden and prognosis: reverse dippers have the worst prognosis while dippers and mild dippers fall into an intermediate risk range. The risk profile of extreme dippers is still debated, and the available data are very conflicting and inconclusive. Starting from this gap of knowledge, we aimed to evaluate, retrospectively, in a cohort of hypertensive subjects, the degree of cardiac involvement in relation to the different nocturnal blood pressure profiles. Methods: We retrospectively evaluated 900 patients with essential hypertension, of whom 510 met our study criteria. We graded the 510 patients in relation to the percentage of reduction in mean systolic blood pressure (SBP) at night-time compared with day-time, considering this as a continuous variable, and then compared the extreme quintiles with each other and with the middle quintile (considered as reference). Results: Patients with less (or no) reduction in nocturnal SBP (reverse dipper) showed a higher level of organ damage and comorbidities. With regard to echocardiographic indexes, patients with maximum nocturnal pressure reduction (extreme dipper) showed a lower level of remodeling and/or impairment of E/e’ ratio, Right Atrium Area, Basal Right Ventricular Diameter, Inferior Vena Cava Average Diameter, and Tricuspidal Anular Plane Systolic Excursion compared also with hypertensive patients with a physiological nocturnal pressure reduction, even after correction for the main confounders. Conclusions: These data suggest that extreme dippers may constitute the subgroup of hypertensive patients with the lowest 24-h pressure load and, therefore, less cardiac remodeling. Blood pressure (BP) has a physiological circadian rhythm known for many decades . The phyA criticism is related to the evidence that the ABPM predictive value depends on the criterion used to classify the circadian rhythm of blood pressure. By using four nocturnal profiles rather than just two (dippers and non-dippers), significant differences emerge: reverse dippers  have the worst prognosis and the higher burden of comorbidities compared to the other three dipping categories ,7,8,9, wStudies that have examined ABPM data with the aim of assessing more closely the presence of differences in cardiac involvement according to the nocturnal pressure profile and, therefore, the nocturnal pressure load of the hypertensive patient, have consistently assessed that the non-dipper profile is associated with increased LV-RWT ,11 and LStarting from this gap of knowledge, we aimed to evaluate retrospectively in a cohort of hypertensive subjects the whole cardiac involvement of extreme dipper hypertensives comparing the level of impairment with the other dipping categories.We retrospectively evaluated 900 patients affected by essential hypertension among those referring to the outpatient clinic of the Division of Internal Medicine and Stroke Care of the University of Palermo, Italy, in the period between 1 January 2016 and 31 March 2020. Among these, we analyzed those for which all data were available from a transthoracic echocardiogram and an ABPM performed, as well as all anamnestic, clinical, and laboratory data essential to the clinical classification of the patient. 510 patients meet these inclusion criteria.-Diagnosis of secondary hypertension;-Record of an acute vascular event  in the six months prior to the date of the evaluation;-Supraventricular arrhythmias ;Clinical history or orthostatic hypotension, autonomic dysfunction or diabetic neuropathy;2;Body mass Index > 35 Kg/mHistory of sleep disturbance (including patients with obstructive sleep apnea) and/or night-workers;Every condition contraindicating the reliability of the ABPM:Exclusion criteria:The initial study procedure included the evaluation of the clinical records with special reference to Body Mass Index (BMI) and the following blood biochemical examinations . Subjects were defined as type 2 diabetics if they had known diabetes treated by diet, oral hypoglycaemic drugs, or insulin.Previous cerebrovascular disease (TIA/ischemic stroke) was assessed by history, specific neurological examination performed by specialists, and hospital or radiological (brain computed tomography or brain magnetic resonance) records of definite previous stroke.Previous coronary heart disease (CHD) was assessed by history, clinical examination, electrocardiogram, and echocardiogram.Essential hypertension was defined on the basis of clinical history, hospital records, and/or antihypertensive treatments taken daily by the individuals. In our study, only subjects with no change in antihypertensive therapy in the last two years have been considered for the analysis. Angiotensin Receptor Blockers (33.1%) and Calcium Channel Blockers (31.5%), was the most commonly used antihypertensive drugs in our sample, followed by ACE-inhibitors (22.4%), beta-blockers (21.9%), diuretics (21.4%), and other drugs  (13.9%).All enrolled patients had performed ABPM in our unit. The methods of performing and reporting the procedure were as follows. ABPM was performed by a TM-2430 Recorder by A & D Company Limited of Tokyo, Japan. This device provides an oscillometric record. The recorders employed in the current study had previously been validated and recommended for clinical use . The mon-dippers: mean reduction in night-time BP between 10 and 20% ;-mild dippers: night-time drop in BP is between 0 and 10%, i.e., a night-day BP ratio > 0.9 and ≤1);-reverse dippers: paradoxical increase in BP during the night (night-day BP ratio > 1);-extreme dippers: night-time reduction in BP is >20% (night-day BP ratio ≤ 0.8);Measurements recorded during the 24 h were stored on a personal computer and screened as follows: a 24 h record was rejected for analysis if more than one-third of the potential day and night measurements were absent or invalid. The ambulatory BP values used for statistical analysis were expressed as 24-h average systolic and diastolic pressures, and 24-h average heart rate. The night/day ratio of BP was calculated as follows: mean nocturnal systolic BP/mean diurnal systolic BP, considering for the analysis the night/day BP ratio as a continuous variable. According to current guidelines,  the fourAll enrolled patients had performed a transthoracic echocardiogram in our unit. The examinations were performed with a GE-Vivid 7 echocardiographic machine with the patient placed supine and in left lateral decubitus by the same experienced echocardiographer and stored digitally for off-line evaluation using a specific program, to be subsequently interpreted by the same echocardiographer who performed the test. All measurements and echocardiographic data were collected and analyzed in accordance with the latest guidelines of the American Society of Echocardiography (ASE) . The par-Left ventricular end-diastolic volume measured in ml (LV-EDV). We considered reference values ranging from 42 and 58.4 mm for men and 37.8 and 52.2 mm for women.-Left ventricular mass and Ejection Fraction . The mass of the left ventricle was calculated in grams, using the following formula: 0.8 (1.04 [(LVIDd + IVS + PWT)3/LVID3]) + 0.6 g. where IVS is the end-diastolic interventricular septal thickness, LVID is the left ventricular end-diastolic diameter, and PWT is the posterior wall end-diastolic thickness. The procedure of our study included indexing of left ventricular mass (LVMI), since this system allows the comparison of ventricular masses of subjects having different body weights. We considered a normal left ventricle indexed mass in case of values between 49 and 115 g/m2 for men and 43 and 95 g/m2 for women. We speak of left ventricular hypertrophy (LVH) when LVMI values exceed 115 g/m2 in men and 95 g/m2 in women and considered normal a LVEF > of 52% for men and >54% for women.-Relative Wall Thickness (RWT).-Left atrium volume (LAV). The volume obtained by measuring atrial areas and diameters was indexed for body surface area (LAVi), and a left atrial volume of up to 34 mL/m2 was considered normal in both genders.-E/A ratio and E/e’ ratio. LV diastolic function was evaluated through the capture of Tissue Doppler images at mitral cusps level, obtained through a two-dimensional apical window of the four cardiac chambers, were used to measure the transmitral flow velocities; this was measured (in m/s) during the peak of early passive diastolic filling  and during the late peak of the diastolic flow due to atrial contraction (A-wave). The E/A ratio was then calculated, which decrypts blood flow from the atria to the ventricles during ventricular diastole and provides information about the atrial contribution to ventricular filling. The lower the ratio, the greater the atrial contribution. The E/A ratio value in a subject with normal diastolic function is between 0.8 and 2, but the correct evaluation of the E/A ratio requires a broader framework of the data and, therefore, it is more correct to consider a normal pattern and three abnormal patterns: altered diastolic release (E/A < 1), pseudonormal pattern  and restrictive pattern (E/A > 2).For the left ventricle:Pulsed tissue Doppler of the mitral annulus was also used to measure the early protodiastolic peak (e’) of the septal and lateral mitral annulus velocities in addition to the lateral tricuspid annulus velocities; all velocities are expressed in cm/sec. The E/e’ ratio was derived using the mean value of e’ of the septal versant lateral side of the mitral valve. An E/e’ ratio less than 8 is typically associated with normal filling pressures, whereas a ratio > 15 indicates increased filling pressures (ventricular diastolic dysfunction). In accordance with the ASE Recommendations for the Evaluation of Left Ventricular Diastolic Function , the fin-Area of the right atrium. According to the Guidelines for the echocardiographic assessment of the right heart in adults [2 regardless of sex.n adults  we consi-Basal diameter of the right ventricle. It was measured at the basal third of the right ventricle and has been considered a normal range between 25–41 mm. The right ventricle is assumed to be dilated if the basal diameter is >41 mm.-Diameter of the inferior vena cava. According to the recommendations, a VCI diameter < 21 mm associated with inspiratory collapse > 50% suggests an atrial pressure of 3 mmHg (range 0–5 mmHg), whereas a VCI diameter > 21 mm with inspiratory collapse < 50% suggests an elevated atrial pressure, approximately 15 mm Hg (range 10–20 mmHg). In indeterminate cases, in which the VCI did not meet the two profiles, an intermediate value of 8 mmHg (range 5–10 mmHg) was considered.-Tricuspid annular plane excursion (TAPSE). It is measured in M-Mode by placing the cursor on the lateral tricuspid annulus from the apical 4-chamber projection. TAPSE quantifies the systolic excursion of the tricuspid annulus along the longitudinal plane thus assessing the efficiency of contraction; it is, therefore, a reliable index of right ventricular systolic function demonstrating a good correlation with other parameters such as myocardial scintigraphy and 2D estimation of right ventricular ejection fraction. The greater the excursion, the better the performance of the right ventricle. A value > 17 mm was considered normal in both genders.-Estimated systolic pulmonary artery pressure. Right ventricular systolic pressure was estimated through the velocity of tricuspid regurgitation using the simplified Bernoulli equation (dP = 4V2), which allows measurement of the right ventricular-atrial pressure gradient. Adding this value to the right atrial pressure estimated by assessing the diameter and respiratory excursions of the inferior vena cava will yield an estimate of pulmonary arterial pressure. The maximum tricuspid regurgitation velocity was considered normal if ≤2.8 m/s, whereas the maximum normal trans-tricuspid gradient was considered for values ≤ 36 mmHg.For the right ventricle:t test for paired data was used to compare differences for both continuous anthropometric data and laboratory variables before and after the intervention. Discrete variables were analyzed using the Chi-square test and Fisher’s exact test, if necessary. One way ANOVA test was performed to determine any statistical differences between the means of the quintiles considered. We added a Multivariate Analysis of Covariance (MANCOVA) to correct to correct the differences observed between the quintiles for the major confounder, the mean values of 24 h SBP. A post hoc analysis with the Bonferroni test was added when necessary to confirm the differences between the groups. We also evaluated the strength of the correlation between the nocturnal BP reduction (Δ mean SBP Day/Night %) as a continuous variable and the echocardiographic parameters studied through the calculation of the Pearson’s Correlation Coefficient. Data were analyzed using Epi Info Software  and SPSS Software version 21.0 . p values less than 0.05 were considered statistically significant.Statistical analysis was performed splitting the population into quintiles with reference to the variable Δ mean SBP Day/Night %, considering it as a continuous variable. The comparison was made between the upper quintile (consisting of the group of extreme dipper subjects), including patients with the most significant nocturnal pressure drop, the lower quintile (consisting of the group of reverse dipper subjects), including patients with less or no nocturnal pressure drop, and the middle quintile, considered the reference quintile (dipper subjects), having a physiological nocturnal reduction in BP. Statistical analysis of quantitative and qualitative data, including descriptive statistics, was used for all items. Continuous variables are presented as mean ± 1 standard deviation where not otherwise specified. The student’s p < 0.0005; have a higher burden of comorbidities such as diabetes (p < 0.0001); impaired renal function (p < 0.0005); COPD (p < 0.0001); anamnestic vascular events (p = 0.033). Extreme dipper hypertensives in our case history have the lowest frequency  of diabetes (p < 0.0001).p = 0.019) and extreme (p < 0.0005), respectively; the lowest diurnal DBP levels: 75.8 ± 11.8 mmHg vs. 80.9 ± 8.6 and 83.1 ± 8.6 for ref (p = 0.001) and extreme (p < 0.0005), respectively. Extreme dipper hypertensives as a result of the increased night-time BP reduction have the highest levels of morning surge for both SBP  and DBP . The three quintiles have superimposable diurnal mean SBP and DBP levels.p values for the main confounder mean 24-h SBP value. From the comparison between the top and bottom quintiles with the middle quintile, our data show that reverse dipper hypertensives have a higher level of myocardial function impairment than all the others, whereas extreme dipper hypertensives seem to show merely no index of more cardiac damage than the other quintiles examined but a lower level of cardiac involvement even in comparison with subjects with normal circadian BP rhythm as witnessed by lower LV diastolic impairment , smaller right atrium size  and a generic lesser RV involvement as can be detected by the smaller size of RV basilar diameter , smaller size of IVC  and higher values of TAPSE .p < 0. 0001), LVMI , Basal RVD  and RA area .p < 0.0001), VCI , TAPSE , E/A .Weaker correlations have been found for the other parameters evaluated: LA Volume Considering a large cohort of 510 subjects with essential hypertension divided into quintiles with reference to the level of nocturnal BP drop, it is possible to highlight significant differences in the epidemiology, clinic, organ damage, level of comorbidity, and finally cardiac involvement.(2)Mild and reverse dipper hypertensives, as already reported by several authors, are those burdened by a worse clinical, laboratory, instrumental, comorbidity, and organ damage profile.(3)Extreme dipper hypertensives, on the contrary, even when compared with the dipper profile  represent the population of subjects with the lowest systemic involvement as well as the lowest level of organ damage.(4)The cardiac involvement during hypertensive disease is different in relation to the nocturnal BP profile, to an extent that seems to go beyond the epidemiological and clinical differences between the groups. A higher nocturnal reduction of SBP seems to be linked to a less marked cardiac involvement;(5)Our data, obtained in groups of subjects with superimposable age, type and mean number of antihypertensive drugs taken daily, duration of hypertensive disease, and day-time BP load, adjusted for the main confounder, 24-h SBP, reaffirms the prognostic relevance of night-time compared with day-time BP.The main findings of our study are the following:One of the undoubted advantages of the ABPM use is the possibility to record and use for clinical and therapeutic purposes the measurements obtained during the night-time. If we split hypertensive patients into only two nocturnal dip categories, i.e., dippers  and non-dippers or mild dippers , what we observe is higher total mortality, cardiovascular morbidity, and a higher level of organ damage for non-dippers ,8, but wp < 0.01) with a trend towards the lower frequency of cardiovascular mortality and cardiovascular events (p: 0.07). Our data, collected in groups of subjects with superimposable age, type and mean number of antihypertensive drugs taken daily, duration of hypertensive disease, and day-time BP load, seem therefore to support the hypothesis that, for some aspects, such as cardiac organ damage, the extreme dipper BP profile is not to be considered an increased risk profile, but a sort of “super-dipper” profile, in which the lower BP load in 24 h due to the greater fall in BP at night can slow down the progression of the disease.Historically, two patterns of circadian BP variability were associated with an increase in cardiovascular risk: those with higher nocturnal BP levels  ,7,8,9,24Our data, which show maximal cardiac involvement in subjects with higher nocturnal BP, are in line with literature ,16,24,29(1)Retrograde transmission of the increased afterload through the dysfunctional LV and thus of the dilated LA associated with a possible increase in pulmonary resistance . This me(2)Peculiar susceptibility of the pulmonary circulation of the hypertensive patient to catecholaminergic stress resulting in prolonged vasoconstriction and overload on the RV .(3)As a consequence of interventricular septum remodeling induced by chronic BP overload, there was a demonstrated direct mechanical transmission of parietal stress from left to right .(4)The activation of bio-humoral mechanisms related to cardiac remodeling in the hypertensive patient  act as much on the left as on the right chambers .Interesting is our data concerning the different involvement linked to the different nocturnal BP profiles, also of the right sections of the heart, wrongly perceived as less affected by the damage due to the chronic BP increase. The relationship between the LV, which is directly influenced by a persistent overload of central BP, and the RV is extremely complex. The architecture and the pattern of contraction of the two ventricles are intrinsically different. The main mechanisms that make global involvement of the four cardiac chambers in the hypertensive patient pathophysiologically plausible are:Our results are partially comparable with those of Tadic et al. , who evaThe reasons explaining how a different level of myocardial organ damage can be associated with a different level of nocturnal blood pressure reduction are partly unknown, but they certainly imply multifactorial mechanisms. One determinant is represented by the different sympathovagal balance and the different levels of adrenergic activation. In addition to contributing to the development, maintenance, and progression of the hypertensive state, sympathetic hyperactivity certainly also participates in the genesis of major complications related to target organ damage in the hypertensive patient. It has been reported that in reverse dippers there is sympathetic hyperactivation  and reduOur study has some limitations: the first is the retrospective design of the study; a prospective study designed ad hoc could more accurately define the degree of organ damage in relation to the maintenance over time of the same nocturnal BP profile. The second is the limited reproducibility of nocturnal BP reduction in different monitoring performed in the same subject ,42. ThisIn conclusion, our study suggests that hypertensive subjects with the greatest reduction in nocturnal BP being the subgroup with the lowest 24-h BP load may have less cardiac remodeling regardless of the duration of the disease, mean 24-h BP levels, and antihypertensive therapy used, reaffirming the prognostic relevance of night-time compared with day-time BP.


Background: Kidney perfusion and oxygenation are two important determinants of kidney graft function. In kidney transplantation, repeated graft hypoperfusion may occur during hip flexion, for example in the sitting position, due to the progressive development of fibrotic tissue around iliac arteries. The aim of this study was to assess the changes in oxygenation and perfusion of kidney grafts during hip flexion and extension using a new functional magnetic resonance imaging (fMRI) protocol.Methods: Nineteen kidney graft recipients prospectively underwent MRI on a 3T scanner including diffusion-weighted, blood oxygenation level dependent (BOLD), and arterial spin labeling sequences in hip positions 0° and >90° before and after intravenous administration of 20 mg furosemide.Results: Unexpectedly, graft perfusion values were significantly higher in flexed compared to neutral hip position. Main diffusion-derived parameters were not affected by hip position. BOLD-derived cortico-medullary R2* ratio was significantly modified during hip flexion suggesting an intrarenal redistribution of the oxygenation in favor of the medulla and to the detriment of the cortex. Furthermore, the increase in medullary oxygenation induced by furosemide was significantly blunted during hip flexion (p < 0.001).Conclusion: Hip flexion has an acute impact on perfusion and tissue oxygenation in kidney grafts. Whether these position-dependent changes affect the long-term function and outcome of kidney transplants needs further investigation. Kidney transplantation is the therapy of choice for patients with end-stage kidney disease conferring survival benefit regardless of graft source . HoweverIn spite of advanced operative techniques, vascular complications after kidney transplantation remain of concern , 6. SurgIntriguingly, lower extremity claudication has been reported in professional cyclists due to kinking of iliac arteries during hip flexion visualized by magnetic resonance (MR) angiography , 9. KinkLittle is known about dynamic perfusion changes in kidney grafts depending on body posture. Kidney grafts typically placed into iliac fossa might be exposed to similar position-dependent perfusion problems. Specifically, kinking or narrowing of the transplant renal or iliac artery may occur during hip flexion due to tethering by adjacent fibrotic tissue leading to iterative hypoperfusion episodes as well as chronic ischemic graft damage and IFTA in the long-term. Considering common sedentary lifestyle majorly in the sitting position, this influence could be significant. Furthermore, pre-existing endovascular lesions in this high-cardiovascular-risk population or subclinical vascular anastomotic problems may have an additional effect. To the best of our knowledge, this question has not been addressed so far.Today, novel functional MR imaging (fMRI) techniques allow for the non-invasive investigation of renal tissue oxygenation, perfusion, and diffusion with the use of blood oxygenation level dependent (BOLD)-MRI, arterial spin labeling (ASL)-MRI, and diffusion-weighted imaging (DWI) .The aim of this prospective interventional study was therefore to assess the influence of hip flexion on kidney graft oxygenation and perfusion using fMRI techniques in kidney transplant recipients. Our hypothesis was that hip flexion >90°  would lead to an instant and temporary reduction of renal tissue oxygenation and perfusion as measured by BOLD-MRI, ASL-MRI, and DWI compared to neutral hip position. To correlate results with vascular anatomy , non-contrast-enhanced time-of-flight (TOF) angiography during hip flexion and duplex-ultrasound scans (DUS) were performed. Additionally, we analyzed the correlation of oxygenation and perfusion changes with clinical parameters.The protocol of this prospective single-center interventional study was approved by the local ethics committee  and conducted in accordance with the Declarations of Helsinki and Istanbul , 15.2 according to Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)-equation and preserved faculties of judgment were eligible.Patients ≥18 years having received a kidney graft ≥6 months ago into the iliac fossa with a stable graft function  and an estimated glomerular filtration rate (eGFR) ≥30 ml/min/1.73 mExclusion criteria were pregnancy; New York Heart Association stage IV dyspnea or orthopnea; acute infection; active neoplasia; surgical intervention, severe trauma, or acute ischemic or thromboembolic event within the preceding 2 months; inability of ipsilateral hip flexion; classical contraindications to MRI; body weight >200 kg; as well as any implanted metallic material without prior 3T-MRI after implantation.From June 2015 through January 2016, 97 consecutive patients were screened at the outpatient University Clinic for Nephrology and Hypertension in Bern, 19 of whom fulfilled eligibility criteria and accepted to participate. Written informed consent was obtained from each participant prior to inclusion. A standardized hydration protocol was followed  and diuretics were held on the study day and the preceding day if considered safe by the treating nephrologist . A lightThere were four study phases during one patient's session: anatomical MRI, DWI, BOLD-MRI, and ASL-MRI were first performed during neutral hip position and then repeated during maximally achievable hip flexion (≥90° from bed level); to account for position-induced confounding effects on the BOLD signal as well as to include a functional test, the same measurements were repeated 10 min after the intravenous administration of 20 mg furosemide  –21. At tMRI data were acquired on a 3.0 T whole body MR Scanner . To facilitate hip flexion >90°, measurements were performed in the lateral decubitus position requiring subject positioning far off-center. Maintenance of hip flexion was assured by an MR-safe belt.*; outcome measure is the transverse relaxation rate R2*  which correlates to tissue oxygen content when confounding factors such as blood volume or hydration state are excluded. Rather than providing absolute values of oxygenation, this technique is especially useful to demonstrate relative changes in response to various interventions .ASL-MRI allows quantitative perfusion measurements using magnetically labeled water as an endogenous diffusible tracer , 23, 24.D and the perfusion fraction FP (2), two repetitions, TR of 3300 ms, TE of 56 ms, slice thickness of 5 mm, matrix of 128 × 128 and FOV of 300 × 300 mm2.DWI allows the quantification of diffusion parameters and microperfusion yielding ADCction FP , 22. DWITOF angiography is an MR technique permitting the visualization of vascular flow without the need for contrast agents. Based on the phenomenon of flow-related enhancement of spins entering into an imaging slice, vascular anatomy can be reconstructed in a three-dimensional view.A maximum of six regions of interest (ROIs) were analyzed in every slice  for each of the four measurements . ROIs were manually defined by the same blinded investigator on images handed out in a random fashion (mixed between patients and study phases). ROIs containing approximately 10 voxels were traced in the medulla and cortex. All images were co-registered facilitating comparable ROI position for all methods. Data were analyzed using in-house custom-scripts written in IDL® and MATLAB®.Serum creatinine was measured by an enzymatic creatinine assay .Doppler studies were performed according to clinical routine in the outpatient clinic of the University clinic for Nephrology and Hypertension in Bern on an Acuson® 2000S device  including graft morphology, urinary outflow, and perfusion .* values, in medullary to cortical R2* ratio (MCR R2*) and in R2* ratio without/with (wo/w) furosemide during hip flexion compared to neutral hip position. Secondary outcomes were the changes in mean ASL perfusion values and mean FP by DWI during hip flexion compared to neutral hip position, the presence of renal transplant/iliac artery kinking visualized by TOF angiography during hip flexion, the correlation of oxygenation  and perfusion changes with the presence of functional kinking, endovascular lesions of transplant renal/iliac artery (evidenced by DUS) and with clinical parameters , as well as correlation of these clinical parameters with functional kinking.The primary outcome was the change in mean medullary and cortical R2*; the diffusion coefficient ADCD; and perfusion value of <7, 4, and 15%, respectively, could be detected in 19 patients on a significance level of 0.05 and 80% statistical power  received an incomplete dose of furosemide due to venous access problems. A mean scanning time per study phase of 15–20 min was met, resulting in an overall measurement time for all four scans <90 min including a break leaving the magnet and the time for furosemide injection. Overall visual image quality was judged good despite unusual lateral decubitus in both positions and intermediate for TOF angiography, however, without performing a formal image quality analysis . BOLD, A* values as a marker of tissue oxygenation level  are shown in * values were significantly higher in the medulla. Medullary R2* decreased significantly after furosemide administration, as reported for native kidneys .To investigate determinants of position-induced oxygenation and perfusion changes, correlation testing was performed between fMRI and selected clinical parameters . To reduce the number of comparisons, only significant changes between positions were tested. Two significant correlations were found:* from neutral to flexed position without furosemide correlated positively with eGFR . This might indicate a stronger cortical R2* increase (oxygenation decrease) during flexion in patients with reduced graft function.First, in BOLD, the change in MCR R2p = 0.041) and transplant vintage  as well as with CNI use after furosemide .Second, in ASL, the increase in cortical perfusion from neutral to flexed position without furosemide correlated negatively and significantly with age  Hip flexion diminishes the increase in oxygenation after furosemide. At last, our study demonstrates for the first time the feasibility and reliability of a multiparametric functional MRI protocol with examination of kidney graft oxygenation and perfusion during two different body positions in a single session.The main findings of this study are the following: (1) hip flexion induces a consistent increase in renal perfusion as measured by ASL; (2) hip flexion causes a redistribution of renal tissue oxygenation with a significant decrease of the medulla/cortex ratio R2Our initial hypothesis was that hip flexion would lead to an instant and temporary reduction of graft perfusion associated with a decrease in graft oxygenation. To our surprise, perfusion values measured by ASL-MRI consistently increased during hip flexion. ASL technique involves the magnetic labeling of inflowing blood with the creation of a subtraction image of the kidney with and without labeling . In modeThe first is an increase in arterial inflow due to the partial obstruction distally to the graft leading to increased flow through the external and internal iliac artery. In line with this hypothesis, maximal hip flexion has been shown to induce a shortening, bending, and twisting of iliac arteries in healthy subjects , 34. In A second hypothesis is that hip flexion causes an increase in arterial blood pressure secondary to a global increase in systemic vascular resistance. Analogously, the squatting position classically adopted by patients with cyanotic heart disease leads to blood pressure elevations by increasing venous return and systemic arterial resistance. Yet, this effect might be restricted to the standing position , 36. HowThe increase in renal perfusion could also represent a counter-regulatory mechanism secondary to the changes in tissue oxygenation. Indeed, the scan order putting BOLD before ASL sequences might explain inverse trends in BOLD and ASL values. Thus, in a recent study, sympathetic stimulation by handgrip exercise has been reported to decrease renal artery flow and increase medullary oxygenation measured by BOLD-MRI. A decreased reabsorptive workload due to reduced distal sodium delivery was hypothesized by the authors .Venous outflow obstruction with backflow to the graft during hip flexion is still another mechanism whereby hip flexion could increase graft perfusion. Venous obstruction might actually explain both the increased renal perfusion and the reduced oxygenation response to furosemide. In this situation, the marked prolonged position change might have overcome the hurdle of venous valves. The hydration protocol inducing a larger plasma volume may have increased this effect. Similarly, the two subjects with ipsilateral graft implantation (with a risk of arterio-venous crossing) showed a particularly reduced perfusion ratio neutral/flexed hip position possibly pointing to outflow obstruction. Importantly, potential changes in arterial inflow may have been masked in this case. Thus, in this view, the positive correlation between age, transplant vintage and CNI use on one hand and the cortical perfusion ratio neutral/flexed hip position on the other may be explained by a reduced arterial component of overall flow. However, this hypothesis remains speculative.The perfusion increase in the cortex and medulla during hip flexion after furosemide is augmented by the observed  lower baseline perfusion values during neutral hip position after furosemide compared to the values before the administration of furosemide. This is in line with previous microelectrode measurements in rats and ASL-MRI measurements in humans , 39.* ratio, suggesting a redistribution of intrarenal tissue oxygenation toward the medulla. The decrease of MCR R2* during hip flexion correlated with lower eGFR values. MCR R2* has previously been defined as the normalized intrarenal oxygen bioavailability . The patients/participants provided their written informed consent to participate in this study.LYM participated in research design, performance of the research, data analysis, and writing of the manuscript. MS participated in research design, performance of the research, data collection, and revision of the manuscript. FN participated in data analysis and the revision of the manuscript. DT participated in data analysis and the revision of the manuscript. GD participated in data analysis. PM contributed the arterial spin labeling–magnetic resonance imaging sequence and participated in the revision of the manuscript. MB participated in the writing and revision of the manuscript. BV participated in the research design, performance of the research, and revision of the manuscript. PV participated in the research design, performance of the research, data collection, data analysis, and revision of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ROC curves demonstrated moderate sensitivity (range of 0.636–0.971) and specificity (range of 0.464–0.889), for LSCI and non-invasive blood pressure measurements. Therefore, no association between diabetic foot ulcer healing and LSCI-measured microcirculation or non-invasive blood pressure measurements was found. The healing tendency of diabetic foot ulcers is difficult to predict based on single measurements using current blood pressure measurements or LSCI.Diagnosis of peripheral artery disease in people with diabetes and a foot ulcer using current non-invasive blood pressure measurements is challenging. Laser speckle contrast imaging (LSCI) is a promising non-invasive technique to measure cutaneous microcirculation. This study investigated the association between microcirculation (measured with both LSCI and non-invasive blood pressure measurement) and healing of diabetic foot ulcers 12 and 26 weeks after measurement. We included sixty-one patients with a diabetic foot ulcer in this prospective, single-center, observational cohort-study. LSCI scans of the foot, ulcer, and ulcer edge were conducted, during baseline and post-occlusion hyperemia. Non-invasive blood pressure measurement included arm, foot, and toe pressures and associated indices. Healing was defined as complete re-epithelialization and scored at 12 and 26 weeks. We found no significant difference between patients with healed or non-healed foot ulcers for both types of measurements ( Diabetes Mellitus is a metabolic disease and its patient population is growing worldwide, with a prevalence of 9.3% of the adults between 20 and 79 years old. A total of 463 million people are living with diabetes . One of Multiple methods are used to identify diabetic foot ulcers that are suspicious to poor healing as a result of PAD. Diagnostic arteriography and non-invasive blood pressure measurements are recommended in guidelines of the International Working Group on the Diabetic Foot (IWGDF) for the treatment of diabetic foot ulcers . IndicatNovel optical imaging techniques such as laser speckle contrast imaging (LSCI) are available to complement the currently used non-invasive blood pressure measurements in people with diabetic foot disease . LSCI isIn a previous study among patients with a diabetic foot ulcer, we demonstrated that LSCI is a stable technique with a high inter- and intra-user reliability . We concThe clinical dataset used for this study was obtained as part of a larger study . This stInclusion criteria were a confirmed diagnosis of type 1 or type 2 diabetes mellitus, and one foot ulcer (defined as break of the skin of the foot that involves at least the epidermis and part of the dermis ). ExclusMeasurements were performed after ulcer debridement, and consisted of first doing LSCI scans, followed by non-invasive blood pressure measurements. LSCI scans were performed of the ulcer foot with a PeriCam PSI NR . Either the plantar or the dorsal side of the foot was scanned, depending on the ulcer location, with the ulcer location to be included in the scan. Perfusion was expressed in perfusion units (PU). During the scan, the patient lay supine on the examination table barefoot. After 5 min, for the patient to get used to the room temperature (kept between an ambient 21–22 degrees), the LSCI scans were acquired. During the scans, three different time periods of interest (TOI) were measured: baseline, biological zero, and post-occlusion hyperemia measurements. The baseline was a measurement in the first stage of the scan when the measured perfusion was stable on visual inspection for 30 s. Subsequently, a cuff around the ankle was inflated to stop blood flow to the foot. During this time the perfusion dropped to the biological zero value of the patient. When the perfusion did not further decrease for 30 s, the biological zero was measured. After this measurement, the ankle cuff was released. The maximum measured blood flow after release of pressure was used as the post-occlusion hyperemia value. During each TOI, different regions of interest (ROI) of the foot were measured . As described in detail in our previous paper [Each patient was classified as non-ischemic, ischemic, or critical-ischemic based on non-invasive blood pressure measurements, following IWGDF criteria ,26. PatiClinical background and different parameters  of the p2 test for all categorical variables. Statistical relevance was considered with a p-value less than 0.05. ROC curves were created and the sensitivity and specificity of different parameters were calculated. The thresholds to calculate positive and negative likelihood ratio (LLR+ and LLR–) were chosen based on the highest combination of both sensitivity and specificity. A LLR– between 0.5–1 or LLR+ between 1–5 indicates no small change, while a LLR– between 0.1–0.5 or LLR+ between 5–10 were considered as moderate. LLR– below 0.1 or LLR+ above 10 were considered as large effect [To investigate differences between the healed and non-healed participants at 12 and 26 weeks, t-tests were conducted for all numerical variables and a Chie effect .A total of 61 patients were included. One patient died during follow up, two patients underwent major amputation, and five patients underwent revascularization. Of the 53 patients included for analysis, 23 (43.4%) healed within 12 weeks, 36 (67.9%) in 26 weeks, while 17 patients (32.1%) did not heal in 26 weeks or received revascularization treatment , Table 1p = 0.925) and also no significant differences between these groups at 26 weeks . We chose to do so, because advanced blood pressure assessment is most important in this group from a clinical perspective, as these are patients for whom diagnosis and prognosis are in a grey area. However, these post hoc analyses did not result in different findings (results not shown); again, no differences were seen in blood pressure measurements between patients who healed and patients who did not heal.With no significant differences found between patients who healed and those who did not heal, we repeated all tests for the group of participants classified as ischemic only (The aim of this study was to investigate the association between foot and ulcer (micro) circulation (measured with both LSCI and non-invasive blood pressure measurements) and healing of diabetic foot ulcers at 12 and 26 weeks. We found no significant differences in any of the measurements between the group of healed and non-healed patients, neither at 12 nor 26 weeks. Positive and negative likelihood ratios showed no or only small effects. In our cohort, both LSCI and non-invasive blood pressure measurements were not useful as a standalone prognostic test for diabetic foot ulcer healing. This result is not in line with the outcomes of a recent systematic review by Forsythe et al.  in whichWhile our study had a different approach in calculating the cut-off values for different non-invasive bedside blood pressure measurement tests, we can still use the results of the studies included in Forsythe et al.  to put tSimilar findings are seen when comparing the found LLR+ and LLR– for toe pressure and TcpO2. Our study found LLR+ and LLR– for toe pressure and TcpO2 of 1.46, 0.40, and 1.24, 0.47, respectively. Other studies reported similar LLR+ and LLR– for toe pressure measurements. For example 1.12, 0.88 ; 1.28, 0When we compared our LLR+ for TcpO2 with other LLR+ values, some studies did find larger effects: LLR+ of 10.03  and 5.14As this is the first study to investigate LLR+ and LLR– for LSCI in relation to diabetic foot ulcer healing, there are no findings for direct comparison. However, in light of the above-mentioned studies, our findings are within the expected range. Despite the advantages of measuring at and around the exact ulcer location, and including both baseline perfusion values and stress-test values, LSCI did not result in improved prognostic likelihood ratios for ulcer healing in this cohort, compared with regular non-invasive blood pressure measurements.The following limitations of this study should be considered. First, the combination of more than one prognostic test may provide more useful information on the probability of healing than a single test or test used in isolation . HoweverSecond, the exclusion of patients that underwent a major amputation can be considered as another limitation. A major amputation could have been considered as endpoint, too, in addition to wound healing. This could be useful for clinicians in order to identify the patients with a higher probability of healing without revascularization to pursue a conservative approach. Furthermore, it could be of importance to identify patients with an unacceptable high risk of a major amputation. For those patients, adequate revascularization should be a priority . TherefoThird, the follow-up period of six months and the use of two measurement moments (at 12 and 26 weeks) dichotomizes healing, rather than using the more detailed time to healing in days or weeks. This dichotomization results in diverse groups, where both short healing times (<4 weeks) and long healing times (>20 weeks) could end up in the same group. In further research it might be better to use time to heal (in weeks) as an endpoint for that study. This provides a better understanding and more useful parameter to obtain meaningful insights into the patients’ healing tendency, since the time needed to heal a chronic diabetic foot ulcers usually varies a lot. We decided to use a different approach in this study for several reasons. First, we wanted to compare our results with previous research . Second,A fourth limitation of this study is that drug use and specific additional treatment of the patient (for example offloading or wound dressings) were not taken into account in this study. Where the first might influence microcirculation measurements, the latter might influence healing outcomes. However, because all patients were treated in the same center and by the same clinicians, clinical decisions were considered similar, and therefore not accounted for in analyses. Furthermore, while some drugs might affect microcirculation, no previous study on prognosis has found an effect of such drugs on likelihood ratios for prognosis . Finally, it is questionable whether we can compare our results with outcomes of previous studies. Although we see comparable results, it is likely that the included populations differ. Whereas in the past, the majority of patients with diabetic foot ulcers had been treated in hospitals, currently only the more complex cases visit hospitals or specialized care centers for diabetic foot ulcers. For future research it would be interesting to compare and validate our findings with more recent studies. No association between healing of diabetic foot ulcers and microcirculation measured with LSCI or non-invasive blood pressure measurements was found. We can conclude that both types of measurements were not useful as a standalone prognostic instrument for diabetic foot ulcer healing.


Diabetic kidney disease (DKD) progresses at different rates among patients with type 2 diabetes mellitus (T2D). Early identification of patients with a higher risk of DKD progression is essential to improve prognosis. Epigenetic modifications, particularly DNA methylation, have been independently implicated in T2D and chronic kidney disease. The current study aimed to determine changes in blood DNA methylation that reflects and predicts DKD progression. C57BL/6 mice were fed a high-fat diet (HFD) from weaning and subclassified into two groups, HFD-1 and HFD-2, according to urinary kidney injury marker KIM-1/creatinine ratios (low vs. high) and histological abnormalities (mild–moderate vs. advanced). DNA methylation profiles were determined by reduced representative bisulfide sequencing (RRBS). Our results confirmed early and established DKD at week 9 and week 32, respectively. At week 32, advanced kidney injury was associated with dysregulation of methylation and demethylation enzymes in the kidney. Blood RRBS revealed 579 and 203 differentially methylated sites (DMS) between HFD-1 and HFD-2 animals at week 32 and week 9, respectively, among which 11 were common. The DMS in blood and kidney at week 32 were both related to organ development, neurogenesis, cell junction, and Wnt signalling, while the DMS in blood at week 9 suggested a specific enrichment of kidney development processes. In conclusion, our data strongly support the implication of early blood DNA methylation modifications and DKD progression in T2D that could be used to improve the disease’s prognostication. Diabetes is a global health concern affecting 422 million people worldwide, the majority of which have Type 2 diabetes (T2D) . T2D is Epigenetic modifications are mechanisms whereby different phenotypes can manifest on the same genetic blueprint, which at least in part explains why diseases progress differently among patients with similar genetic backgrounds. Changes in the pattern of DNA methylation, the most studied epigenetic mechanism, have been associated with the development of chronic kidney disease ,8. PatieWith respect to the role of DNA methylation in DKD, the majority of evidence to date has been in Type 1 diabetes (T1D) populations ,10. Althg for 10 min. For a subset of week 32 animals, blood samples were also collected at week 9 from tail vein for DNA methylation analysis. A 24-h urine sample was collected 2 days before cull using metabolic cages. Blood and urine samples were stored at −20 °C until analysis.The study was approved by the Animal Ethics Committee of the Northern Sydney Local Health District (RESP/15/22 and RESP/18/108). All studies were performed in accordance with the relevant guidelines and regulations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. C57BL/6 mice (3 weeks) were fed a high-fat diet  or standard rodent chow . We have previously demonstrated that animals fed a HFD demonstrate glucose intolerance and insulin resistance from 9 weeks . The HOMThe pathologic paradigm for kidney disease progression is advancing tubular injury and interstitial fibrosis . The addThe plasma levels of triglyceride (TG) were measured using Roche triglyceride reagent GPO-PAP (Roche Life Science) and the levels of non-esterified fatty acid (NEFA) were measured using Wako HR Series NEFA-HR(2) kit . Blood insulin and urinary albumin concentration were measured using Ultra-Sensitive Mouse Insulin ELISA and Mouse Albumin ELISA Kits , respectively. Urinary KIM-1 was measured using a Mouse KIM-1 ELISA Kit . The concentrations of urinary and blood creatinine were determined using Urinary and Blood Creatinine Assays, respectively . All assays were performed according to the manufacturers’ instructions.® SYBR® Green Primers Predesigned primers . Gene expression was standardized to β-actin mRNA and log-transformed. Primers’ sequences are as previously published [Total RNA of kidney tissues was isolated using RNeasy Plus Mini Kit  according to the manufacturer’s instructions. The purified total RNA was used as a template to generate the first-strand cDNA using the First Strand cDNA Synthesis Kit . The amplicons of target genes were amplified using KiCqStartublished .Proteins (20 μg) were electrophoresed and electroblotted onto Hybond nitrocellulose membranes , which were then incubated with a primary antibody at 4◦C overnight. The primary antibodies used for immunoblotting included manganese superoxide dismutase  and KIM-1 . α-Tub  was used as the housekeeping protein. Subsequently, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody . The immunoblots were developed by adding Luminata Western HRP Substrates  to the membrane and exposed for an appropriate duration using ImageQuant LAS 4000 . ImageJ  was used for densitometric analyses.® RRBS Methyl-Seq system. Reads  were assessed using FastQC v.0.11.8 [p-values and adjusted p-values  of each pair-wise comparison were calculated based on M-values using the limma package [https://biit.cs.ut.ee/gprofiler/gost, accessed on 8 August 2021) for Gene set enrichment analysis. Blood DNA methylation profiling was undertaken on the same animals at week 9 and week 32 and common differentially methylated CpGs were detected. Pathway enrichment in blood at week 32 was correlated with that in the kidney.DNA was extracted from the blood and kidney of the same animals using a DNeasy Blood and Tissue Kit . Reduced Representation Bisulfite Sequencing (RRBS) DNA library preparation was performed by the Australian Genome Research Facility . The RRBS library was produced using the NuGEN Ovationv.0.11.8 , then trv.0.11.8 . Furtherv.0.11.8  allowed v.0.11.8 , with dev.0.11.8 . p-value package . Beta va package , which wFormalin-fixed kidneys were sectioned at 4 μm thickness and stained for Hematoxylin and Eosin (H&E) and Pico-Sirius Red (PSR). The slides were assessed using a light microscope . The degree of glomerulosclerosis was scored in twenty glomeruli per slide in a blinded manner. PSR images were used to score glomerular and tubular injury, as well as to estimate interstitial fibrosis by Image J software .t-tests and one-way ANOVA followed by Fishers Least Significant Difference (LSD) posthoc test were used for statistical analysis. Data are presented as mean ± SEM. p < 0.05 was considered significant.Unpaired p < 0.001, p < 0.001), while insulin resistance index was also significantly elevated (p < 0.05)  , suggestp < 0.05, p < 0.001), UKCR and plasma creatinine were also significantly elevated . At week 9, the mRNA expression of the oxidative stress marker NAPDH oxidase (NOX)2 was significantly increased (p < 0.001). This was coupled with higher mRNA expression of antioxidant enzymes including superoxide oxidase  and catalase (p < 0.05). No change was found in the expression of inflammatory markers monocyte chemoattractant protein (MCP)-1 and tumour necrosis factor (TNF)α (p < 0.001). MCP1 was also significantly elevated at week 32, suggesting renal inflammation (p < 0.001).At week 9, UACR was significantly increased in HFD-fed mice (r (TNF)α B. In conp < 0.001, p < 0.01) or HFD-1 (p < 0.05)  and tubulointerstitial fibrosis (p < 0.05) in comparison to the control (p < 0.001). In comparison, the kidney of HFD-1 mice showed no significant changes in glomerular and interstitial fibrosis and less severe tubular injury, confirming milder DKD.As described in the methods section, HFD-fed animals were then subclassified into HFD-2 and HFD-1 based on UKCR. UKCR correlated positively with UACR ( < 0.05) B. In par control C. Tubulap < 0.001 and p < 0.05, respectively, Body weight, fat mass, blood lipid, and glucose tolerance were similar between HFD-2 and HFD-1 mice despite different DKD stages at week 32 , thus exp < 0.05, p < 0.05, p < 0.05). In contrast, TET2 and TET3 were upregulated only in the kidney of HFD-1 group . Overall, the data suggest that DNA methylation machinery was differentially modulated depending on the level of kidney injury. Consistently, kidney DNA methylation profiling by RRBS suggests an enrichment of genes involved in the TGF-β signalling pathway, which is well-known as a key mediator of tissue fibrosis , a by-product of DNA demethylation, was significantly increased in HFD-1 group  and late phases (week 32), suggesting that these abnormal DNA methylations had occurred long before advanced kidney injury developed and persisted across the course of DKD progression. Among these, the most significant CpG corresponds to Fibroblast growth factors (FGF) 22, which was hypermethylated in the rapidly progressing group. Multiple FGFs have been shown to be elevated in patients with chronic kidney disease, including FGF21 and FGF23 ,47. The 40, it may also mediate the brain–kidney axis that was enriched in animals with accelerated DKD progression.Among the eleven differentially methylated genes, Angiotensin II receptor (AT1R) type 1b was of particular interest, as AT1R type 1a and 1b are important receptors of angiotensin, playing important roles in the regulation of blood pressure via the renin-angiotensin system (RAS). Renal expression of AT1R increases in diabetic patients with chronic kidney disease and is suppressed by AT1R blockers . HypometIn conclusion, our study identified early differences in blood DNA methylation between animals with Type 2 diabetes that subsequently developed mild or advanced kidney injury. Further studies are required to validate these changes in a larger cohort of animals as well as in diabetic patients with progressive DKD. In addition, gene expression studies are also needed to determine whether these epigenic changes drive kidney-specific transcriptional changes.This section is not mandatory but may be added if there are patents resulting from the work reported in this manuscript.