Cardiovascular disease (CVD) is the most common cause of death in patients with native and post-transplant chronic kidney disease (CKD). To identify new biomarkers of vascular injury and inflammation, we analyzed the proteome of plasma and circulating extracellular vesicles (EVs) in native and post-transplant CKD patients utilizing an aptamer-based assay. Proteins of angiogenesis were significantly higher in native and post-transplant CKD patients versus healthy controls. Ingenuity pathway analysis (IPA) indicated Ephrin receptor signaling, serine biosynthesis, and transforming growth factor-β as the top pathways activated in both CKD groups. Pro-inflammatory proteins were significantly higher only in the EVs of native CKD patients. IPA indicated acute phase response signaling, insulin-like growth factor-1, tumor necrosis factor-α, and interleukin-6 pathway activation. These data indicate that pathways of angiogenesis and inflammation are activated in CKD patients’ plasma and EVs, respectively. The pathways common in both native and post-transplant CKD may signal similar mechanisms of CVD. CKD is associated with a high burden of morbidity in the form of end stage kidney disease (ESKD) requiring dialysis or transplantation2. Furthermore, patients with CKD are at significantly increased risk of death from cardiovascular disease (CVD)4. In fact, CKD patients are 20 times more likely to die from CVD than they are of reaching ESKD4. The association between CKD and CVD is only partially explained by the high prevalence of traditional CVD risk factors (such as diabetes and hypertension) in CKD6. Multiple studies have shown that CKD, itself, is an independent and powerful predictor of cardiovascular events and mortality10. Hence, it has been postulated that “non-traditional” risk factors, such as inflammation, oxidative stress, and endothelial dysfunction contribute to the increased risk of CVD and mortality in CKD15. However, the underlying pathophysiology of CVD in CKD remains incompletely understood.Approximately one in 10 individuals has chronic kidney disease (CKD) rendering CKD one of the most common diseases worldwide18. Of interest, several studies have confirmed the limited predictive value of the traditional CVD risk factors in post-kidney transplantation patients21. Importantly, reduced kidney function is an independent predictor of CVD in post-kidney transplant patients, much like it is prior to transplantation23. Collectively, the literature suggests that the persistent risk of CVD post-kidney transplantation may be due to the same “non-traditional” CVD risk factors that affect patients with native kidney CKD. Endothelial dysfunction has been reported in patients with CKD post-kidney transplantation by our group and others26. Furthermore, in post-kidney transplantation patients, endothelial dysfunction has been shown to correlate with biomarkers of inflammation26. As such, endothelial dysfunction may represent a common underlying mechanism of CVD in CKD patients, pre- and post-kidney transplantation.After kidney transplantation, the risk of CVD is significantly reduced, and yet CVD remains the most common cause of death and graft loss post-kidney transplantation14. We isolated EVs by ultracentrifugation, and then measured the EV proteome with a SOMAscan assay. The SOMAscan assay uses aptamers to quantify > 1300 different proteins, and it can detect the proteins down to the femtomolar range. EVs are sub-micrometer-sized membrane bound particles that are actively shed from cells in response to activation, injury, and apoptosis28. Given the small quantity of protein contained in the EV fraction of plasma, the high sensitivity of the SOMAscan provides a useful method for analyzing the EV proteome. One surprising result of our previous study was that several proteins that were expected to be soluble were enriched in the EV fraction. This indicates that some proteins may preferentially segregate with the soluble or with the EV fractions of plasma, and that there may be value in examining both fractions. Hence, in the current study, we hypothesized that CKD patients, pre- and post-kidney transplantation, may share a similar inflammatory profile, the biomarkers of which correlate with endothelial dysfunction. We sought to characterize the inflammatory profile of the proteome of patients with CKD of the native kidneys and of the post-transplant kidneys compared to healthy controls utilizing the SOMAscan assay. Considering the potential differences between the plasma and EV fractions, we evaluated the proteome of both.In order to develop new therapies for the prevention of CVD in CKD patients, biomarkers are needed that can accurately predict or reflect vascular injury in this population. A challenge in searching for new plasma biomarkers is that abundant plasma proteins, such as albumin, make it difficult to detect low abundance proteins by standard proteomic methods. To detect low abundance plasma proteins one can use assays designed to detect specific analytes  or array-based assays. One can also use various methods to isolate or enrich specific fractions of the plasma proteome. We recently used both of these approaches to analyze plasma samples from CKD patients14. The ‘parent’ study included 30 healthy subjects, 30 patients with stage 3 or 4 CKD, and 30 patients post kidney transplantation with stage 3 or 4 CKD. The detailed recruitment procedures for the ‘parent’ study have been published14. Briefly, healthy subjects were recruited by public advertisement; the only exclusion criteria being pregnancy or breastfeeding. Patients with CKD were recruited from the CKD clinic and kidney transplant recipients were recruited from the transplant clinic . Individuals with CKD and kidney transplant recipients were considered eligible for participation if they were at least 18 years of age, had stage 3 or 4 CKD with a CKD-EPI estimated glomerular filtration equation (eGFR) of 20–59 mL/min/1.73m229, and were able to give informed consent. In addition, kidney transplant recipients were required to be taking maintenance immunosuppression consisting of tacrolimus, mycophenolate mofetil, and corticosteroids. CKD patients and kidney transplant recipients were excluded if they were pregnant or breastfeeding, had uncontrolled hypertension, body mass index (BMI) ≥ 40 kg/m2, life expectancy < 1 year , hospitalizations within the last 3 months, or active infection on antibiotic therapy. For individuals with CKD of their native kidneys, history of immunosuppressive therapy in the last year was an additional exclusion criterion. The study was approved by the Colorado Multiple Institutional Review Board and all the study procedures were conducted in accordance with the federal human subjects regulations. The study procedures were conducted during 1 visit at the Clinical and Translational Research Center (CTRC) at the University of Colorado Anschutz Medical Campus after patients provided informed consent. During this visit, the study team collected the pre-determined clinical variables including blood pressure and body weight and height, obtained a blood draw for clinical labs and EDTA plasma . In order to validate our findings, we pursued an additional cohort of patients with CKD stage 3b and 4. We randomly selected samples from subjects who had enrolled to participate in 2 currently ongoing clinical trials. The first (NCT03223883) is recruiting patients with stage 3b and 4 CKD to evaluate whether curcumin improves vascular and cognitive function in patients in patients with CKD. In addition to stage 3b and 4 CKD, subjects must be 45–75 years old and have BMI < 35 kg/m2 and able to give informed consent. Exclusion criteria include: consuming a diet rich in curcumin or taking curcumin supplements in the past 12 months, pregnant, breastfeeding, or unwilling to use adequate birth control, uncontrolled hypertension, life expectancy < 1 year , hospitalization within the last 3 months, active infection or antibiotic therapy, or received immunosuppressive therapy within the last year. The second trial (NCT03597568) is designed to evaluate if resveratrol supplementation improves vascular function in patients with stage 3 CKD and diabetes mellitus (DM). The inclusion criteria are age 45–80 years, stage 3 CKD, diagnosis of type 2 DM, use of angiotensin converting enzyme inhibitor or angiotensin II receptor blocker for > 3 month prior to the study, and the ability to give informed consent. The exclusion criteria include: > 2 glasses/day red wine and/or taking resveratrol or vitamin C supplement in the past 12 months, BMI > 40 kg/m2, pregnant, breastfeeding, or unwilling to use adequate birth control, uncontrolled hypertension, uncontrolled DM, life expectancy < 1 year , hospitalization within the last 3 months, active infection or antibiotic therapy, or received immunosuppressive therapy within the last year. Of note, all the participating subjects had signed a consent agreeing to the use of their samples in future research and only samples from the baseline visit were utilized.This is a sub-analysis of a previously published pilot study2. Blood pressure was obtained via an automated cuff after 10 min of rest at the beginning of the study visit. Serum creatinine and albumin to creatinine ratio (ACR) were measured by the clinical lab. eGFR was calculated based on the CKD-EPI formula29 and urinary ACR was reported as mg/g.Race/ethnicity were based on patient self-reporting. DM status was defined either as history of DM according , current treatment with oral hypoglycemic agents or with insulin, or fasting glucose ≥ 126 mg/dL. BMI was calculated as kg/m30. Briefly, EDTA plasma samples were and stored at − 80 °C until used. To separate the EVs, the samples were thawed in a 37 °C water bath and were centrifuged at 400× g for 15 min at 4 °C. The supernatants were then collected and the volume recorded. Next, 250 µl of each sample was placed in a polyallomer tube. The plasma was centrifuged at 20,000× g for 2.5 h using a Beckman XL-80 ultracentrifuge with a sw55ti rotor. Braking was applied at the end of the centrifugation. EV buffer  was used to re-suspend the pellets to a volume of 100 µl  by pipetting up and down six times31. To characterize the EVs, we performed Western blot analysis on a protein lysate generated from the EV fraction of 3 mL of plasma from a CKD patient31. Protein samples were separated by electrophoresis on 10% Criterion TGX gels  and transferred to Immulon P membrane . The lysates were then probed using antibodies to CD63 , CD81 , TSG101 , and Calnexin . A protein lysate generated from A549 cells  was used as a positive control for the Calnexin blot. The antibodies were detected with appropriate horse radish peroxidase (HRP)-conjugated secondary antibodies and electrochemiluminescence reagent . Bands were seen in the blots probed for CD63, CD81, and TSG101  BCA protein concentration analysis. The Somalogic SOMAscan assay was then used to analyze the proteome in an EDTA plasma sample and an EV sample for each included subject at the University of Colorado Genomics and Microarray Core. The SOMAscan assay is a multiplexed proteomics assay that uses aptamers (single stranded DNA molecules) selected to bind specific protein targets. The bound proteins are then quantified. This assay is advantageous for analyzing complex samples because it has high very high sensitivity and wide dynamic range, and high abundance proteins in the sample  do not obscure the detection of low abundance proteins. The assay panel utilized detects > 1300 proteins. There are some limitations to this assay, however, including cross-reactivity of the aptamers with non-target proteinsLevel of Ephrin B2 in the plasma was determined via enzyme-linked immunosorbent assay (ELISA) by a commercially available kit . Samples were run as described in the kit manual through the reagent B incubation step and subsequent washing. ELISA Amplification system (ELAST)  was inserted to the reaction system according to the manufacturer's instructions. The ELAST streptavidin-HRP concentrate was diluted 1/750. Selected analytes were also measured using Meso Scale Discovery (MSD) assays . MSD uses electrochemiluminescence to detect proteins with high sensitivity. We used multiplex (VEGF-A and VEGF-D) and singleplex (CFD) assays according to the manufacturer’s instructions. Briefly, plates were coated and incubated overnight at 4 °C with continuous rotary shaking at 700 rpm. Plasma was either diluted two-fold and incubated for two hours (VEGF-A and VEGF-D) or 1000-fold and incubated for one hour (CFD). Analyte intensities were captured with the MESO QuickPlex SQ 120 instrument and analyzed with MSD Discovery Workbench, v4 software .33 which we felt was important considering the pilot nature of our pursuit. First, we identified the proteins that were differentially expressed in the native and post-transplant CKD patients versus the healthy controls. Then, we ranked these proteins in order of magnitude and significance prior to conducting IPA analysis34. Proteins were ranked and included in the IPA if they were found to have differential expression > 1 or < 1 with a p value of < 0.05. Subsequently, we evaluated the potential correlation between urinary albumin/creatinine ratio (uACR) and the overlapping proteins . uACR was evaluated considering it is well known marker for increased CVD risk and mortality in addition to CKD progression36. We opted to evaluate these correlations in the combined group of subjects defined as native kidney CKD and post-kidney transplant CKD consistent with our hypothesis that CVD risk may share similar non-traditional pathways in both groups. Statistical analysis was carried out using SAS version 9.4 .Descriptive statistics are reported by study group as N (%) for categorical variables and mean (SD) or median (IQR) for continuous variables. Fisher’s exact test was used to test for differences in categorical variables between the 3 different conditions, while the Kruskal–Wallis nonparametric test was used for continuous variables. In reporting individual protein expression, significance was set at a p value < 0.0002 in order to account for multiple comparisons. Ingenuity pathway analysis (IPA) was utilized as the bioinformatics approach for the proteomics analysis. IPA allows for the functional interpretation of data such as ours14. We identified 16 healthy controls, 16 CKD patients (stage 3 or 4), and 16 kidney transplant recipients with CKD who had residual EDTA plasma samples at a volume adequate for the analysis. The clinical characteristics of the included subjects are shown in Table The clinical characteristics for all the participants in the ‘parent’ study have been published elsewhereNine subjects in each group had adequate sample remaining for EV analysis. The clinical characteristics for the subjects with adequate sample for the EV analysis are shown in Supplementary Table The SOMAscan assay revealed significant differences in the plasma proteomes of the three study groups - B2, Eph family receptor interacting proteins (ephrin) A2 and A5, and vascular endothelial growth factor (VEGF). Increased levels of additional proteins linked to angiogenesis were identified, including insulin-like growth factor-binding protein (IGFBP-6), secreted modular calcium-binding protein 1 (SMOC1), and endostatin. These data are summarized in Table In comparing the post-transplant CKD patients to the healthy controls, we identified a pattern similar to that observed in native kidney CKD. The heat map in Fig. After identifying the proteins that differed in the CKD patients versus the healthy controls and those that differed in the post-transplant CKD patients versus the healthy controls, we evaluated whether any of these proteins overlapped between the CKD patients and the post-transplant CKD patients. Of note, this analysis included all the proteins noted to be significantly different in comparing the CKD patients with the healthy controls or the post-transplant CKD patients with the healthy controls . Figure uACR is a clinical indicator of vascular disease. In evaluating the 44 proteins that overlapped in between the native kidney CKD and post-kidney transplant CKD groups, the following were found to correlate significantly with uACR: desmocollin-2 (DSC-2), cystatin C, fatty acid-binding protein, liver-type (FABPL), regenerating islet-derived protein 4 (REG-4), IGFBP-6, CD59, ephrin-A2, A4, and A5, and EFN-B2. These data are shown in Table 37. We previously reported that the size and number of medium-sized (100–1000 nm) endothelial EVs was the same in samples from patients with CKD, transplant CKD, and healthy controls, but we did not analyze the protein content of the EVs14. The SOMAscan tissue assay was used to analyze the proteomes of the EVs from the three patient groups. We observed fewer significant differences between the circulating EV of the 3 study groups than we observed in the plasma samples. These data are shown in Supplemental Fig. Circulating EVs are derived from cells. They carry proteins from the parent cell, and plasma proteins can adhere to their surfaceProteins known to be increased in patients with CKD were found to be elevated in the EV fraction of the CKD patients as compared to the healthy controls, including cystatin C and β2-microglobulin. In addition, the renin precursor protein (REN), which originates in the kidney, was significantly higher in patients with CKD compared to healthy controls. Figure Figure We next evaluated which proteins were differentially expressed in EVs from both CKD patients and the post-transplant CKD patients. As in the plasma analysis, we included all the proteins noted to be significantly different in comparing the CKD patients’ EVs or the post-transplant CKD EVs with the healthy control EVs . Eighteen differentially expressed proteins were found to overlap between the CKD and post-transplant CKD groups including biomarkers of kidney disease  and biomarkers of inflammation , and TNFRSF-1B). These data are illustrated in the VENN diagram in Fig. Of the 18 eV proteins increased in both the native kidney CKD and post-kidney transplant CKD groups, β2-microglobulin and cystatin C were found to correlate significantly with uACR. These data are shown in Table p value = 0.0057). High sensitivity MSD assays are available for CFD, VEGF-A, and VEGF-D, three of the analytes measured by the SOMAscan assay. We therefore used MSD assays to confirm the SOMAscan results for these proteins, and we also validated these findings using samples from a second cohort of CKD patients. VEGF-D and CFD levels were significantly elevated in the CKD samples when measured with MSD, similar to the results obtained by SOMAscan. Similarly, the levels of both proteins were significantly higher in the validation cohort compared to healthy controls. Although VEGF-A levels were higher in the CKD samples than in the healthy control samples by SOMAscan, the levels of VEGF-A measured via MSD in both CKD cohorts were not significantly different from the healthy controls. These data are shown in Fig. Ephrin B2 was measured via ELISA and was detectable in 2 samples in the healthy controls, 6 samples for the CKD samples, and in 11 samples of the validation cohort. Mean(SD) were 9.5(35.9), 14.5(37.1), and 66.4(114.8) pg/mL in the healthy controls, CKD samples, and CKD validation cohort respectively  in order to identify common pathways associated with increased risk of CVD in both patient groups, as the increased risk of CVD persists post kidney transplantation. In addition, the inclusion of patients with post-transplant CKD also allowed us to examine whether markers of inflammation in CKD are affected by treatment with standard immunosuppressive drugs. This approach identified plasma proteins that were differentially and significantly expressed in plasma of patients with native and post-transplant CKD compared to healthy controls. Furthermore, EV proteins were identified in both native kidney CKD and post-transplant CKD. Pathway analysis revealed significant activation of angiogenic and inflammatory pathways in the native and post-transplant CKD as compared to the healthy controls. Many of the identified proteins were significantly correlated with eGFR, and several proteins correlated with albuminuria, which is often used as a marker of microvascular damage. We confirmed the elevations in several analytes including Ephrin B2 ligand (EFN-B2), VEGF-D, and CFD in CKD using ELISA and MSD (a different high sensitivity assay). We also verified that these proteins were elevated in plasma samples from a second cohort of patients with CKD. We had previously described dysregulation of the alternative pathway of complement in this group of subjectsExamination of the EV proteome revealed that this fraction contained an almost completely different subset of proteins than that seen in whole plasma. By analyzing circulating EVs we were able to identify additional pathways that were differentially activated in CKD patients compared to healthy controls. There was a prominent pro-inflammatory profile in the EV proteome of CKD patients, including markers of T-helper cell activation, complement, and TNF pathways activation in both native and post-transplant CKD groups as compared to the healthy controls. Importantly, several biomarkers of kidney disease known to be produced in excess by injured kidneys were only detected in the EVs, including β2-microglobulin, and the renin precursor protein. These findings suggest that the analyzed circulating EVs, at least partially, originated in the kidney. As such, some of the identified proteins could potentially predict CKD progression. Alternatively, our findings may reflect a pathogenic or protective role in kidney disease progression.38 that culminates in the activation of pro-angiogenic growth factors (such as VEGFs and angiopoietins) and their receptors. Vascular injury is also associated with increased vascular permeability, migration of endothelial cells to the site of injury, extracellular matrix remodeling, and ultimately the budding of new vessels40. Several proteins were detected systemically, that signal activated angiogenesis in CKD patients (both native kidney and post-transplant), the most prominent of which belong to the ephrin receptor family. The ephrin receptors consist of the largest family of receptor tyrosine kinases41; at least 16 members divided into two classes, A (ephrin A1–A10) and B (ephrin B1–B6)42. These proteins require cell to cell interaction in order to bind their membrane-associated ligands; the ephrins43. There are 5 ephrin A ligands that bind promiscuously to all ten ephrin A receptors and 3 ephrin B ligands (EFN-B) that interact with the ephrin B receptors42. To our knowledge, this is the first report of altered levels of ephrin ligands/Eph receptors in CKD patients. Considering the data above, ephrin B2 ligand (EFN-B2) is of particular interest. EFN-B2 is induced in endothelial cells with angiogenic activation including in response to VEGF46. In adults, EFN-B2 is upregulated at sites of neovascularization, such as in tumors and wounds48. In addition to promoting capillary network formation and sprouting angiogenesis, EFN-B2 stimulation in endothelial cells is known to promote inflammatory cell adhesion, migration, and chemotaxis50, effects that may promote atherosclerotic vascular disease.This analysis identified several novel biomarkers of angiogenesis in patients with CKD. In adult organisms, angiogenesis, the formation of blood vessels from preexisting vasculature, is an adaptive mechanism in response to hypoxia and vascular injury51, a ubiquitous pro-inflammatory transcription factor that plays an important role in atherosclerosis52. As such, the increased levels of EFN-B2 may reflect underlying inflammation and atherosclerosis. Consistent with a role in atherosclerosis, EFN-B2 is upregulated in human atherosclerotic plaques and expressed in endothelial cells at sites of arteriolosclerosis in mice54. In addition, and some data suggest it interacts with EphB2 receptor on monocytes thus contributing to the release of pro-inflammatory cytokines54. Other members of the ephrin ligand/receptor family may also play a role in atherosclerosis, as EFN-B1 and ephrin B2 expression is increased in human carotid artery atherosclerotic plaques53. It is possible that the higher detectable levels of EFN-B2 are a biomarker of vascular inflammation and atherosclerotic disease in CKD. We identified a significant correlation between many of the plasma and EV pro-angiogenic proteins with uACR, a surrogate marker of endothelial dysfunction, CVD, and mortality in patients with CKD35. Considering the known role of the identified pro-angiogenic pathways in CVD and the identified correlation with uACR, we suspect that the proteins identified via this analysis relate to the increased risk of CVD in patients with CKD. Certainly, data from animal models of CKD indicate that experimental uremia associates with dysfunctional angiogenesis55. Alternatively, some of the proangiogenic biomarkers detected are known to promote angiogenesis in a manner that may improve tissue perfusion in models of vascular disease. For example, VEGF-D is significantly associated with mortality in patients with CAD56. However, gene transfer of human VEGF-D has shown improved cardiac perfusion in animal models and may be of use in cases of refractory angina, stent restenosis, and peripheral vascular disease. Thus, it is plausible that the detected pathways herein may be protective. Longitudinal studies will be needed to determine whether the identified proteins are predictive of CVD. Further experiments will be also required to determine whether the pathways activated in CKD patients are functionally important to the increased risk of CVD and other systemic complications.In addition to hypoxia, there is also evidence to suggest that inflammation induces EFN-B2. Most pertinent to our discussion, the EFN-B2 promoter is responsive to nuclear factor-κB (NF-κB)58. In addition, several studies have implicated these pathways in other glomerular diseases59. VEGF-A, for example, is highly expressed in the podocyte and has been shown to play an important role in the formation and preservation of a functional filtration barrier. Additionally, the dysregulation of VEGF-A results in glomerular disease characterized by proteinuria60. Of note, while VEGF-A was noted to be increased in CKD subjects versus healthy controls, we were unable to reproduce this in the validation cohort. This may be due to higher sensitivity of the originally utilized assay (SOMAscan). EFN-B2 reverse signaling has been shown in response to fibrotic kidney injury and is believed to play a protective role against capillary rarefaction and fibrosis61. Not surprisingly, our findings indicated significant activation of the TGF-β pathway. This was common to both groups of CKD patients, native and post-transplant. Chronic induction of TGF-β is known to cause extracellular matrix accumulation62 with resultant glomerular and tubulointerstitial fibrosis65 and hence is believed to play an important role in the progression of kidney disease. As it pertains to our findings here, TGF-β induction is reported to occur in response to tissue hypoxia and in association with the angiogenic response66. Some have advocated that in the presence of hypoxia the balance between the pro-angiogenic response and the pro-fibrotic response is tipped so that the pro-fibrotic response is more prominent67. Hence, the pro-angiogenic profile we have identified may in fact be a reflection of the pathological processes in the diseased kidney. It is unknown whether pro-angiogenic pathways contribute to kidney disease in post-transplant CKD and perhaps this is a valid path of investigation in the future.It is important to recognize that dysregulated angiogenesis is known to play a role in certain kidney diseases, most notably diabetic kidney disease68. Endostatin is a fragment of collagen XVIII that is highly expressed in the renal glomeruli and pertitubular capillaries and is formed during extracellular remodeling69. Thus, in addition to its anti-angiogenic effects, endostatin may be a biomarker of fibrosis. Indeed, higher levels of plasma endostatin have been linked kidney function decline in patients with type 2 DM69.Lastly, while the majority of detected proteins in this analysis are proangiogenic, some are actually anti-angiogenic. For example, we noted significantly higher levels of endostatin, a protein that has been shown to inhibit pathological angiogenesis and may be a target of therapy in the treatment of cancerOne limitation of this study is the small number of patients and our data should be considered hypothesis generating. Future studies can focus on the panel protein biomarkers and pathways identified in this study to validate these findings in larger patient cohorts. CKD is also caused by many different underlying systemic diseases. A larger number of samples will be required to determine whether there are protein biomarkers specific to particular causes of CKD. We were also unable to evaluate whether any of these novel biomarkers predict hard outcomes and future studies will need to examine whether these biomarkers predict CVD or CKD progression. The SOMAscan assay measures a large panel of proteins with very high sensitivity and specificity, but currently it is cost prohibitive to perform it as a high throughput assay. Nevertheless, our approach overcomes several barriers that have hampered biomarker studies in CKD. The use of an aptamer-based array prevented high abundance plasma proteins from obscuring the lower abundance biomarkers. It also allowed us to detect circulating EV proteins with high sensitivity, as EV proteins represent only a small fraction of the protein contained in a plasma sample.In summary, we have used an aptamer-based proteomics array to identify pathways that are activated in patients with CKD. We analyzed plasma samples as well as isolated EVs, and we identified discrete groups of proteins that are altered in each of these sample types. Our findings indicate activated angiogenesis and inflammatory pathways, raising the possibility that these pathways could represent therapeutic targets. Furthermore, some of these proteins correlated with albuminuria, a biomarker of CVD and kidney disease progression. Future work will be needed to confirm these findings larger cohorts of patients, and to determine whether any of these candidate markers predict CVD and/or CKD progression.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


Overall, these results highlight the importance of the A2AR in AF and suggest that the evaluation of this receptor in PBMCs may be potentially be useful in monitoring disease severity and the efficacy of pharmacological treatments in AF patients.Atrial fibrillation (AF) is the most common form of cardiac arrhythmia seen in clinical practice. While some clinical parameters may predict the transition from paroxysmal to persistent AF, the molecular mechanisms behind the AF perpetuation are poorly understood. Thus, oxidative stress, calcium overload and inflammation, among others, are believed to be involved in AF-induced atrial remodelling. Interestingly, adenosine and its receptors have also been related to AF development and perpetuation. Here, we investigated the expression of adenosine A2A receptor (A2AR) both in right atrium biopsies and peripheral blood mononuclear cells (PBMCs) from non-dilated sinus rhythm (ndSR), dilated sinus rhythm (dSR) and AF patients. In addition, plasma adenosine content and adenosine deaminase (ADA) activity in these subjects were also determined. Our results revealed increased A2AR expression in the right atrium from AF patients, as previously described. Interestingly, increased levels of adenosine content and reduced ADA activity in plasma from AF patients were detected. An increase was observed when A2AR expression was assessed in PBMCs from AF subjects. Importantly, a positive correlation ( Atrial fibrillation (AF) is one of the most common cardiac arrhythmias, which is currently thought to affect approximately 2% of the world population. Its prevalence increases with age, affecting around 0.14% of people under 49 years old and increasing to 10-17% in people aged 80 or older . AF epis1R, A2AR, A2BR and A3R, some of these have been related to AF physiopathology. For instance, it has been demonstrated that A2AR density is increased in AF patients, leading to an abnormal calcium handling in the cardiomyocyte and triggering heart disfunctions [2AR overexpression and activation by selective agonists may favour the occurrence of cardiac arrhythmias [1R by intrinsic adenosine modifies atrial electrophysiology and promotes AF [Adenosine is a purine nucleoside generated mostly by ATP catabolism and regulated by adenosine deaminase (ADA), adenosine kinase (ADK) and S-adenosyl-l-homocysteine hydrolase (SAHase) . Adenosiunctions ,17. Therhythmias . Conversmotes AF . Accordimotes AF .2ARs both in right atrium biopsies and peripheral blood mononuclear cells (PBMCs) from AF patients. In addition, the adenosine levels and adenosine deaminase (ADA) activity in plasma from the same patients were evaluated.Here, we set out to assess the status of the adenosinergic system in AF by analysing the expression of A2AR in right atrial tissue [2AR density (p < 0.0001) in the right atrium from AF patients when compared to non-AF subjects  (p = 0.0207) increase in A2AR mRNA expression in right atrial tissue from these patients was also found  A,B  = 3.753) (p = 0.1312) but not AF  = 44.66) (p = 0.0202) between ADA activity and adenosine content within ndSR and AF patients was found  A. Yet, nt not AF A. Subseq= 44.66) B. Indeedas found C. Collec2AR density in PBMCs from these patients. To this end, we used a fluorescent A2AR antagonist, namely MRS7396 [2AR density in PBMCs. First, we validated the specificity of MRS7396 A2AR staining in HEK293-A2ARSNAP cells by displacing its binding with ZM241385, a selective A2AR antagonist  when compared to both ndSR and dSR patients  = 23.55)  = 14.64)  A. Next, = 14.64) B.2AR density in PBMCs and in right atrium of the same patients was observed   was found  A, thus ias found B,C. Finaobserved D,E. Over2ARs play a key role in coronary flow and force generation, they have been also associated with some cardiopathological conditions, such as the genesis of arrhythmias [2AR revealed once again an increased receptor density and RNA expression in the in right atrium biopsies from AF patients, in agreement with previous work [2AR in PMBCs from AF patients was increased when compared to ndSR and dSR subjects, thus, mirroring that observed in right atrium. In addition, the adenosine levels in plasma from AF patients were also increased and correlated with A2AR density in PBMCs and in right atrium. Collectively, these results point towards the existence of a potential cardiac-peripheral connection with the adenosinergic system in both health and disease and highlight the importance of the A2AR in AF.While cardiac Ahythmias ,22. Hereous work . Interes2AR regulates PBMCs function [2AR have been found in lymphocytes from patients with immunological diseases, including systemic lupus erythematosus [2AR density in lymphocytes also increased in patients with coronary artery disease [2AR and PBMCs is now revealed for AF, although the specific mechanism underlying that remodelling of A2AR has not yet been determined. Moreover, the altered levels of adenosine and ADA activity in AF raises the question of whether the increased adenosine levels in AF are a cause or consequence of the disease. If peripheric adenosine augmentation triggers AF, then it might be cogitated if a reduction in ADA activity is behind AF pathophysiology. Interestingly, in CHF a reduction in ADA and ADA2 activity has been reported [2ARs remodelling. Importantly, signalling through Afunction , and incematosus , amyotroematosus , multiplematosus  and rheuematosus . In addi disease  and chro disease . This lireported , thus, areported . Yet, he2AR expression. Elevated adenosine levels and A2AR expression observed in patients with AF supports the notion that excessive A2AR activation contributes to a higher incidence of spontaneous calcium release-induced electrical activity and irregular beating reported in atrial myocytes from patients with AF [2AR expression? On one hand, a possible explanation is a positive regulatory loop between adenosine and A2AR where adenosine is released to the environment due to excessive and abnormal beating and a consequence of the energy spent on it. This release would induce a positive turnover of A2AR expression, which could be necessary to keep the heart in a fast-beating condition. On the other hand, adenosine levels are probably a consequence of AF, which leave us the possibility that A2AR enhanced expression is the first step on the AF progression. This leads to abnormal heart beating due to a disturbance in calcium homeostasis and triggering higher adenosine levels. This would suggest a causative correlation, where the A2AR expression is altered prior to the adenosine content. In addition, there could be other, and even more complex, reasons behind the increased A2AR density and adenosine levels in AF, which might promote both simultaneously, without any apparently relationship between them. Interestingly, we found a correlation between adenosine levels and A with AF ,31, but 1R-dependent anti-adrenergic vasodilatory effects will protect the myocardium, whereas still benefited by the improved perfusion pressure. Thus, further research is needed to reveal the mechanism underlying the precise interplay between A2AR and A1R in AF. Overall, endogenous adenosine plays a key role on central cardiovascular regulation and it has profound modulatory effects through systemic mechanisms of autonomic regulation . Thus, i2AR , rabbit anti-α-actinin , horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG  and HRP-conjugated goat anti-rabbit IgG .The following reagents were used: adenosine deaminase , MRS7396 ,33, ZM24Human whole blood Samples (4 mL) from 28 non-dilated sinus rhythm (ndSR), 24 dilated sinusal rhythm (dSR) and 27 atrial fibrillation (AF) patients   were colg at 4 °C for 30 min. The membranes were dispersed in 50 mM Tris HCl (pH 7.4) and 10 mM MgCl2, washed, and resuspended in the same medium as described previously [Right atrial samples were pulverized in liquid nitrogen congelation and sonicated  in 500 μL of ice-cold 10 mM Tris HCl, pH 7.4 buffer containing a protease inhibitor cocktail . The sonicated tissue was further homogenized using a Polytron  for three periods of 10 s each. The homogenate was centrifuged at 12,000 × eviously . Protein®-LFP polyvinylidene difluoride (PVDF) membranes  using the Trans-Blot®TurboTM transfer system  at 200 mA/membrane for 30 min. PVDF membranes were blocked with 5% (wt/vol) dry non-fat milk in phosphate-buffered saline  containing 0.05% Tween-20 (PBS-T) during 1h at 20 °C before being immunoblotted using mouse anti-A2AR (0.5 µg/mL) and rabbit anti-α-actinin (0.5 µg/mL) antibodies in blocking solution overnight at 4 °C. PVDF membranes were washed with PBS-T three times (5 min each) before incubation with either a HRP-conjugated rabbit anti-mouse IgG  or HRP-conjugated goat anti-rabbit IgG  in blocking solution at 20 °C during 2 h. After washing the PVDF membranes with PBS-T three times (5 min each), the immunoreactive bands were developed using a chemiluminescent detection kit (Thermo Fisher Scientific) and detected with an Amersham Imager 600 .Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was performed using 10% polyacrylamide gels. Proteins were transferred to Hybond® ND-1000 UV-Vis spectrophotometer . Reverse transcription was performed using 0.5–1 µg of total RNA obtained from each sample using StaRT Reverse Transcription Kit  according to the manufacturer’s instructions. To quantify A2AR mRNA expression, we used TaqMan single tube assay probe . The TBP (TATA-box binding protein) probe  was used to normalize mRNA expression from both PBMC’s and heart samples. Reaction conditions were done following manufacturer’s instructions in a 384-well plate . The reaction was carried out in a total volume of 10 µL per reaction. The reaction mix included 5 µL TaqMAn Fast Advanced Master Mix, 0.5 µL TaqMan Assay Probe, 3.5 µL of Nuclease Free Water and 1µL of cDNA template  and were done in 7900HT Advanced Real-Time PCR instrument (Applied Biosystems). Amplification protocol started with 95 °C for 10 min, 42 cycles of 95 °C (10 sec) and 60 °C (30 sec), a final cycle of 95 °C (10 sec), and finishing with a 60 °C (30 sec) step. The results are presented relative to those for the housekeeping gene TBP using the ΔCq method. Data were analysed with Expression Suit Software v1.2 .Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density centrifugation using Lymphoprep . PBMCs were stored at −80 °C in Triazol until use. Cell RNA was purified using RNeasy Plus Universal Mini Kit . Heart tissue was pulverized upon liquid nitrogen congelation and sonicated in 100 µL of Tris (pH 7.4) buffer and stored with 1ml of TriPure reagent  at −80 °C. Subsequently, the RNA was separated from DNA/proteins using TriPure  and following manufacturer’s instructions. RNA yield was determined using a NanoDrop−1. Mobile phase was A) Ultrapure water with 0.1% HCOOH and B) Acetonitrile with 0.1 %HCOOH. The gradient program was as follows : , , , , , , . Mass spectrometry detection was performed by using the multiple reaction monitoring (MRM) mode using the following parameters: ion spray voltage, +5500 V; source temperature, 600 °C; curtain gas, 20 psi; ion source gas 1 and gas 2, 50, and 20 respectively; collision-activated dissociation gas, High; entrance potential, (+/-)10 V. The MRM transitions for adenosine were 268.3/136.1  for quantitative purposes and 268.3/119.1  for confirmation purposes dA-d3 was used as internal standard with a transition 230.9/115 . The calibration curve was constructed with adenosine standard solutions between 0.03–8 ng/mL diluted in water TCA30%. Linear regression was adjusted (1/x or 1/x2) to have accuracies between 80-120% for all the adenosine standards. Analyst 1.6.2 Software was used for data acquisition and MultiQuant 3.0.1 for data processing both from ABSciex .Quantitative analysis of ribonucleosides  was carried out as previously described ,35,36. IPlasma from each patient’s blood was isolated by density centrifugation using Lymphoprep  and stored at −80 °C until use. ADA was determined using a commercial colorimetric ADA assay kit  and following the manufacture’s indications. Adding the reagents of the kit you produce a colorimetric reaction based on quinone dye. Therefore, ADA activity in each plasma sample (5 µL) was determined in triplicate by measuring the absorbance at 550 nm using a POLARstar Omega plate reader .2ARSNAP construct [v/v) fetal bovine plasma at 37 °C and 5% CO2.Human embryonic kidney (HEK)-293T stable cell line permanently expressing the Aonstruct  were mai2ARSNAP expressing HEK-293T cells and human PBMCs (4 × 105 cells) were incubated with 50 nM MRS7396 in presence or absence of 100 nM ZM241385  in 300 µL of complete DMEM, or RPMI, respectively, under constant rotation  during 16 h at 4 °C. Subsequently, cells were centrifuged at 500× g for 5 min at 4 °C and rinsed once with ice-cold Hank’s balanced salt solution  and immediately used for flow cytometric measurements or confocal imaging.A4 cells (events) were analysed using a BD FACSCanto II flow cytometer  with excitation at 488 nm and emission at 647 nm. Samples were maintained in the dark and at 4 °C during the analysis to avoid photobleaching and ligand dissociation. PBMCc were selected by size and shape, excluding the death cells, triplets and doublets, while the mean fluorescence intensity per event was obtained in the FL-1 channel in log mode. Data were collected using FacsDiva Software v6 1.3  and the analysis was made using the FlowJo v10.1 software .Flow cytometry measurements were conducted as previously described . In brieFor confocal imaging cells were seeded with DMEM or RPMI medium containing 1 μg/mL of Hoechst 33342 (Thermo Fisher Scientific) in an 8-well treated plate  for 20 min at 4 °C. Confocal images were captured using ZEISS microscope . Analysis was done with ZEN software . p < 0.05. The number of samples/subjects (n) in each experimental condition is indicated in the corresponding figure legend. Outliers were assessed by the ROUT method [p < 0.05). Comparisons among experimental groups was performed by Student’s t test or one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons post-hoc test using GraphPad Prism 9, as indicated. The correlation coefficients (r) were calculated using the Pearson two-tailed correlation test.Data are represented as mean ± standard error of mean (SEM) with statistical significance set at T method , thus, s


Inertial sensors (IS) enable the kinematic analysis of human motion with fewer logistical limitations than the silver standard optoelectronic motion capture (MOCAP) system. However, there are no data on the validity of IS for perturbation training and during the performance of dance. The aim of this present study was to determine the concurrent validity of IS in the analysis of kinematic data during slip and trip-like perturbations and during the performance of dance. Seven IS and the MOCAP system were simultaneously used to capture the reactive response and dance movements of fifteen healthy young participants (Age: 18–35 years). Bland Altman (BA) plots, root mean square errors (RMSE), Pearson’s correlation coefficients (R), and intraclass correlation coefficients (ICC) were used to compare kinematic variables of interest between the two systems for absolute equivalency and accuracy. Limits of agreements (LOA) of the BA plots ranged from −0.23 to 0.56 and −0.21 to 0.43 for slip and trip stability variables, respectively. The RMSE for slip and trip stabilities were from 0.11 to 0.20 and 0.11 to 0.16, respectively. For the joint mobility in dance, LOA varied from −6.98–18.54, while RMSE ranged from 1.90 to 13.06. Comparison of IS and optoelectronic MOCAP system for reactive balance and body segmental kinematics revealed that R varied from 0.59 to 0.81 and from 0.47 to 0.85 while ICC was from 0.50 to 0.72 and 0.45 to 0.84 respectively for slip–trip perturbations and dance. Results of moderate to high concurrent validity of IS and MOCAP systems. These results were consistent with results from similar studies. This suggests that IS are valid tools to quantitatively analyze reactive balance and mobility kinematics during slip–trip perturbation and the performance of dance at any location outside, including the laboratory, clinical and home settings. Postural instability and falls are common occurrences in older adults and stroke survivors resulting in numerous injuries and hospitalizations ,2,3. VolInertial sensors (IS) provide a new direction of motion analysis for the smooth and effective evaluation of the comprehensive kinematics of human motion ,14. InerProper gait in terms of appropriate stepping strategy forms the basis of human locomotion and fall prevention due to slips and trips ,19. The Reactive balance strategies elicited upon exposure to an external support surface perturbation are common mechanisms of determining fall risk. These strategies form the basis for implementing perturbation training programs, to reduce fall incidence in healthy and pathological populations ,19. ThesAdditionally, dance as a multisensory modality has the ability to improve gait, balance, and reduce fall risk . HoweverThus, the aim of the present study was to determine the concurrent validity of IS in measuring kinematic variables of balance and mobility related to fall risk during treadmill-based perturbation (slip and trip) and dancing. Perturbations of varying intensities and dancing with variable song paces were provided to participants. Then, the variables of IS were compared to those from the MOCAP system (silver standard) for equivalence and accuracy. It was hypothesized that there would be reasonably good to excellent agreement of values of the variables analyzed in both systems.Fifteen (15) young participants (18–35 years) without prior exposure to treadmill-based perturbation, and without any involvement in dance routines for at least 1 year (via prescreening interview) consented to participate in this study. Participants responded to a general health questionnaire to ascertain their health status. All participants with recent surgery or any musculoskeletal or cardiovascular conditions, which may impede successful participation in the study were excluded from the study. This laboratory-based study was approved by the institutional review board (IRB) of the University of Illinois at Chicago and conducted in the Cognitive Motor and Balance Rehabilitation Laboratory.Kinematic data were simultaneously obtained with an 8-camera MOCAP system  and with 7 inertial sensors . The 8-camera optoelectronic MOCAP system was able to sense infra-red rays emitted by 29 reflective markers (Helen Hayes marker set), which were attached to various segments or joints of participants such as the sacrum, thighs, shanks, knee, and ankle joints to generate both position, segment, joint angles, and COM state. Concurrently, the 7 IS were attached to the sacral region, thighs, shanks, and middle toe of each foot. These sensors are infused with tri-axial accelerometers, magnetometer (±1.9 g), and gyroscopes (±2000°/s). As such, these sensors can capture the position, joint angles (estimated from segment angles), and COM state of human motion . To gene2), while the displacement increased from 0.21 m for level 1 to 0.36 m for level 5, and their peak velocity increased from 0.31 m/s to 0.86 m/s.For concurrent validity, participants underwent 2 activities, which included treadmill-based stance perturbation tests (slips and trips) of varying intensities from levels 1 to 5, and dance movements with three songs of varying paces from slow to fast. For the treadmill perturbation, the displacement, velocity, and acceleration were the same, but with opposite directions for slip and trip. Across all levels, the acceleration was always constant , participants experienced a series of slip-like forward and trip-like backward stance perturbations. The slip and trip perturbations provided to participants were of different intensities with 10–35% (average of 21.5%) increments in intensity (belt velocity) from the lowest (level 1) to the highest (level 5). The recovery foot is the one that undergoes a compensatory step post-perturbation while the perturbed foot is the one that undergoes slipping or tripping by sliding either forward or backward on the belt of the treadmill. Postural stability variables of interest were mainly stability and step length at touchdown (TD) of the unperturbed limb negotiating a compensatory or recovery due to perturbation. These variables for each system were then matched for equivalence and accuracy. Since stability is usually determined after the first reactive stepping foot touchdown during slip and trip perturbations, stabilities of the lower limb at touchdown were the only measures considered for analysis. Thus, the outcome variables of interest for slip and trip perturbation validity test were stabilities at TD relative to the recovery foot and perturbed foot and step lengths at TD (step length_TD) of the recovery foot.Reactive balance was evaluated with the Stance Perturbation Test. For this task, which was performed on an ActiveStepFor this activity, participants dance to three songs of slow , medium (130 bpm), and fast pace (138 bpm). A protocol from a previous study  was implData collected were processed by digitization via MOCAP and IS software to generate initial COM, gait, and segment angle data. Then, further processing was completed using a customized MATLAB code to generate COM state, step length of the recovery foot touchdown post-perturbation, and segment angles of the lower limbs. These data were then used to calculate stability values during the perturbation test.To ensure that the coordinate systems were the same for the two systems, the calibration procedure was first used to determine the coordinated systems of IS and MOCAP systems. For the MOCAP calibration, an L-Frame was placed on the ground, the long arm determined the x-axis (or AP direction), the short arm determined the y-axis , the line vertical to both arms was the z-axis. For the IS, the north-south direction always represented the x-axis while the east-west direction represented the y-axis. To ensure both coordinate systems are identical, we placed the long arm of the L-Frame in the north-south direction and the short arm in the east–west direction for MOCAP calibration.The COM state was calculated relative to the edge of BOS in the anterior–posterior (AP) direction, and then normalized by foot length or proposed  and implproposed .t-test performed to determine any significant differences between variables of IS and MOCAP systems. A concurrent validity analysis via SPSS  was conducted to determine the similarity between the outcome variables for the two systems during the performance of each task. Initially, Bland Altman (BA) plots and the determination of root mean square error (RMSE) were used to ascertain the agreements of stability and mobility variables between the two motion capture systems. Secondly, correlation coefficient (R) and intra-class correlation (ICC) were used to determine the agreement of variables between the IS and MOCAP systems. To demonstrate the effect of the song on body segment, correlations between each song pace and each segmental angle excursion were determined. The significant level of p-values, α, was set at 0.05.The Kolmogorov–Smirnov test was used to check for normality of data with a paired All data were found to be normally distributed.t-tests showed no significant differences between the two systems (p > 0.05). Validity analysis of reactive balance variables during treadmill-based slip and trip perturbations depicted moderate to high level of similarity of values obtained from the MOCAP and IS systems. This was demonstrated by the Bland Altman plots, Limits of Agreements and the calculation of RMSE, shown in p < 0.05).For slip perturbations, analysis conducted with 75 data points from 15 subjects included data of five slip–trip levels or intensities from each participant. For all reactive balance and stability variables, paired p > 0.05). Bland Altman plots, Limits of Agreements and RMSE are shown in p < 0.05). The R, and ICC scores for mobility variables included segmental angle excursions are shown in For the dance task, there were 42 data points obtained from 14 participants, which consisted of data for three dance songs from each participant. Data for one participant were excluded because of noise in data created by distortions in the IS system. Results conducted by paired t-tests for mobility variables during the dance movements with the three paces of the song showed no significant differences of variables between the two motion capture systems (p < 0.05). With the medium-paced song, there were significant correlations with Lt shank and foot excursions, while dance the fast-paced song demonstrated significant correlations with all segmental angle excursions (p < 0.05), except for the Lt foot excursion, see For each song, there was good concurrent validity of joint mobility values between the two motion capture systems because the R and ICC positively correlated with all segment angles. Dancing with slow-paced song correlated highly with Rt shank and Lt foot angle excursions , and in dance performance. In this study, the reactive balance and mobility measures (stability and step length at touchdown) during slip and trip perturbations were highly comparable in both systems. Equally, the mobility measures (segment angle excursions) during dancing were highly equivalent in both systems. This implies that the hypothesis of similarity and reasonably good agreement (with relatively close to 95% LOA) of values analyzed between both systems was adequately realized in this study. It should be noted that by advanced processing, joint angles could be extrapolated from the segment angles (raw data extracted from both systems) presented in the results.The optoelectronic MOCAP system, as a silver standard tool for motion analysis, has been the most reliable tool predominantly utilized in clinical studies related to reactive balance control in varying populations in the laboratory setting ,19,31. ALaboratory-based studies on fall prevention have examined reactive balance control during slip and trip perturbations with key variables such as stability and joint angles at touchdown ,33. ThesDance is an emerging alternative intervention that is generally accepted and enjoyed by every population group and is increasingly being implemented to improve gait, balance, and mobility or range of motion ,39. AddiThis study demonstrated the concurrent validity of IS by showing that analysis of reactive balance and mobility outcome measures during slip and trip perturbations assessed via the Stance Perturbation Test could be performed with IS with high accuracies, like the MOCAP systems. Though there is scanty evidence on studies on dance training, which have been conducted with MOCAP system, this study showed that studies on dance therapy could be also conducted to determine its effects on postural control and mobility measures with IS. With the determination of the moderate to high validity of IS for assessing balance kinematics during slip and trip-like perturbations and dancing, there is increased assurance for clinicians or researchers to evaluate the progress of home therapy interventions. Additionally, with the current momentum shifting towards the translation of the benefits of research into the daily activities of healthy aging, and neurological-impaired individuals such as stroke, the increased validity of IS will pave the way for the increased home or community-based research and employ the portable IS tool to conduct an objective assessment of balance and fall risk of participants during perturbation training and dance training.


For five decades, blood pressure lowering treatment has been recommended for patients with hypertension (currently defined as blood pressure of ≥140/90 mm Hg). In the past 20 years, guidelines for treatment began incorporating predicted absolute cardiovascular disease risk (predicted risk) and reducing blood pressure thresholds. The blood pressure threshold at which to start treatment has become a secondary consideration in some countries. We aimed to provide descriptive data to assess the relative importance of blood pressure thresholds versus predicted risk on the subsequent rate of cardiovascular disease to inform treatment decisions.In this English population-based cohort study, we used linked data from the Clinical Practice Research Datalink (CPRD) GOLD, Hospital Episode Statistics Admitted Patient Care, and the Office for National Statistics mortality data, and area-based deprivation indices (Townsend scores). Eligible patients were aged 30–79 years on Jan 1, 2011 (cohort entry date) and could be linked to hospital, mortality, and deprivation data. Patients were followed up until death, end of CPRD follow-up, or Nov 31, 2018. We examined three outcomes: cardiovascular disease, markers of potential target organ damage, and incident dementia without a known cause. The rate of each outcome was estimated and stratified by systolic blood pressure and predicted 10-year risk of cardiovascular disease .Between Jan 1, 2011, and Nov 31, 2018, 1 098 991 patients were included in the cohort and followed up for a median of 4·3 years . Median age at entry was 52 years (IQR 42–62) and 629 711 (57·3%) patients were female. There were 51 996 cardiovascular disease events and the overall rate of cardiovascular disease was 11·2 per 1000 person-years (95% CI 11·1–11·3). Median QRISK2 10-year predicted risk was 4·6% (IQR 1·4–12·0) and mean systolic blood pressure before cohort entry was 129·1 mm Hg (SD 15·7). Within strata of predicted risk, the effect of increasing systolic blood pressure on outcomes was small. For example, in the group with 10·0–19·9% predicted risk, rates of all cardiovascular disease rose from 20·1 to 23·6 per 1000 person-years between systolic blood pressures less than 110 mm Hg and 180 and higher mm Hg. But among patients with systolic blood pressure 140·0–149·9 mm Hg, rates rose from 6·9 to 52·3 per 1000 person-years between those with less than 10·0% risk and those with 30·0% or higher predicted risk.For a wide range of blood pressures, the rate of cardiovascular disease and effectiveness of blood pressure drug treatment was mainly determined by predicted risk, with blood pressure thresholds 140/90 mm Hg or 160/100 mm Hg—ubiquitous in most countries—adding little useful information. When medium-term predicted risk is low, there is no urgency to initiate drug treatment, allowing time to attempt non-pharmacological blood pressure reduction.National Institute for Health Research. Blood pressure lowering treatment has been a cornerstone of cardiovascular disease prevention for more than 50 years and for several decades most international guidelines have recommended blood pressure lowering treatment for patients with hypertension (blood pressure ≥140/90 mm Hg).Two major changes have occurred. First, most guidelines now incorporate predicted risk to guide treatment decisions in patients with mild hypertension (blood pressures between 130/80 mm Hg or 140/90 mm Hg and 159/99 mm Hg).Evidence before this studyA 2021, individual patient meta-analysis showed that a 5 mm Hg reduction in systolic blood pressure led to a reduction in risk of cardiovascular disease  across the blood pressure spectrum. This finding indicates that patients with the highest predicted risk of cardiovascular disease, irrespective of baseline blood pressure, would benefit most from blood pressure lowering treatment.We reviewed global blood pressure treatment guidelines for the primary prevention of cardiovascular disease. We found that absolute predicted cardiovascular risk is now commonly used, in addition to blood pressure, to help guide treatment. However, there is still a focus on hypertension, blood pressure cutoffs, and on targets and control of blood pressure as the key goals to lower cardiovascular disease incidence.Given the results of the meta-analysis, guideline committees and clinicians need a clear understanding of the relative importance of blood pressure and predicted risk on the subsequent rate of cardiovascular disease and target organ damage.Added value of this studyOur study indicates that emphasis on blood pressure targets and control should not detract from focus on an overall reduction in predicted risk. Substantial gains could be made to reduce the burden of cardiovascular disease by treating a large group of patients with high predicted risk.Implications of all the available evidenceThis study and the evidence to date indicate that a shift is required to focus away from blood pressure cutoffs and goals, and towards viewing blood pressure in the context of predicted risk for optimal cardiovascular disease prevention.Second, in the USA, the blood pressure lowering treatment threshold was reduced in 2017, from 140/90 mm Hg to 130/80 mm Hg, on the basis of findings from the SPRINT trial.This study aims to provide descriptive data to show the relative importance of blood pressure thresholds and predicted risk on the subsequent rate of cardiovascular disease, to guide the use of blood pressure lowering drugs in a contemporary population.In this population-based cohort study, we used linked data from the UK Clinical Practice Research Datalink (CPRD) GOLD, Hospital Episode Statistics (HES) Admitted Patient Care,Eligible patients were aged 30–79 years on Jan 1, 2011 (cohort entry date) and could be linked to hospital, mortality, and deprivation data. This age range was chosen because patients younger than 30 years would rarely meet treatment eligibility criteria and those older than 79 years required careful consideration and were more likely to be treated outside of guideline recommendations. The cohort entry date was chosen to allow sufficient follow-up to accrue outcome events. Patients with follow-up of less than 1 year before cohort entry, an existing diagnosis of cardiovascular disease or target organ damage, fewer than two blood pressure measures before the start of follow-up, or with the most up-to-date blood pressure measure taken more than 5 years before cohort entry were excluded . PatientEthical approval was granted by the London School of Hygiene & Tropical Medicine Ethics Committee (reference 22955). Study protocol approval was granted by the Independent Scientific Advisory Committee (protocol 19_113).codes to define outcomes. We used coded chronic kidney disease, which in the UK has been incentivised to be recorded as part of the Quality and Outcomes Framework in primary care, rather than estimated glomerular filtration rate (eGFR), because coding implies the general practitioner is aware of the condition and will incorporate it into clinical decision making.This study examined three outcomes: cardiovascular disease; markers of potential target organ damage; and incident dementia without a known cause. Cardiovascular disease included any record of coronary heart disease , atherosclerotic cerebrovascular disease , peripheral arterial disease, and heart failure. Markers of potential target organ damage, which encompass damage to the brain, heart, eyes, and kidneys, are thought to be caused by high blood pressure. This outcome included haemorrhagic stroke and chronic kidney disease . The outcome of incident dementia without a known cause excluded alcohol or drug-induced dementia, Parkinson's disease dementia, Pick's disease, and Huntington's disease. Outcome data were extracted from CPRD, HES and ONS, with the exception of chronic kidney disease which was only extracted from the CPRD database. We used Read and International Classification of Diseases-10 QRISK bundle (version 2.0). QRISK2 scores were categorised into prespecified groups  for 10-year predicted risk. The algorithm to calculate QRISK2 was designed to replace missing data with the same values as the online calculator to obtain a score for all patients. Prescription of blood pressure lowering treatment was assessed at cohort entry on the basis of the most recent ongoing prescription. Rheumatoid arthritis was included as part of the QRISK2 algorithm, but other inflammatory diseases associated with cardiovascular disease were not included.Systolic and diastolic blood pressures were extracted from CPRD primary care records. For analysis, the mean of the last two blood pressure readings within the past 5 years was categorised into prespecified 10 mm Hg bands . Blood pressures extracted for this study were from treated and untreated patients. 10-year predicted risk was measured using QRISK2vs <60 years), and diabetes status at cohort entry . We did not undertake regression analysis or adjust for confounding factors; our analysis was descriptive because the aim was to show differences in rates of outcome for patients in strata to reflect the clinical context in which decisions about treatment are made.The rate of each outcome  was estimated and stratified by systolic blood pressure and predicted risk of cardiovascular disease. In secondary analyses, rates were stratified by blood pressure treatment use at cohort entry , sex, age  and low predicted risk (<10%) and compared with those with normal systolic blood pressure (<140 mm Hg) and high predicted risk (≥20%).We additionally conducted a prespecified analysis of diastolic blood pressure in 10 mm Hg bands.2 (the most recent measure before cohort entry). The need for repeated measures of blood pressure can introduce survivor bias or restrict to those who visit the general practitioner frequently; thus, in sensitivity analysis we used the most recent measure of blood pressure.As coded chronic kidney disease underestimates kidney disease prevalence in primary care, we conducted a sensitivity analysis of chronic kidney disease, defined by an eGFR of less than 60 mL/min per 1·73 mThe funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.Between Jan 1, 2011, and Nov 31, 2018, 1 098 991 patients were included in the cohort and followed up for a median of 4·3 years . Of 2 671 067 patients assessed for eligibility, 1 572 076 were ineligible because they were younger than 30 years or older than 79 years; did not have a blood pressure record; or had fewer than two blood pressure readings, cardiovascular disease, or target organ damage before cohort entry . There wThe rate of outcomes during follow-up, across the blood pressure spectrum and stratified by predicted risk is shown in With the exception of haemorrhagic stroke, patients with low predicted risk (<10%) and high systolic blood pressure (≥160 mm Hg) have strikingly lower rates of outcomes than patients with lower blood pressure (<140 mm Hg) and high predicted risk ≥20%; . For haeFor the composite cardiovascular disease outcome (heart failure and dementia), J-shaped curves were observed which were accentuated in the highest predicted risk group ≥30·0%; .The numbers and proportion of events occurring according to blood pressure and predicted 10-year cardiovascular risk are described in the 209 896 (19·1%) of 1 098 991 patients were using blood pressure lowering medication at cohort entry . There were no substantive differences in rates by predicted risk or systolic blood pressure among patients given blood pressure lowering drugs at cohort entry and those not given such drugs, except for chronic kidney disease, which showed higher rates in patients prescribed blood pressure lowering drugs .vs ≥60), the older age group retained the J-shaped curves observed in the main analysis  patients with diabetes at cohort entry had similar rates of outcomes to those without diabetes . At most2) was substantially higher than the rate measured by coded chronic kidney disease in the main analysis. However, the pattern was similar between the coded and eGFR methods across varying systolic blood pressures and predicted cardiovascular disease risk  at all levels of predicted risk in our study, indicating a need for careful management. This finding is particularly important for young patients (<40 years), whose long-term risk of target organ damage might be high if untreated. At systolic blood pressures lower than 120 mm Hg, the increases in rates of all cardiovascular disease, heart failure, and peripheral arterial disease shown in our study are similar to other observational studies, in which the increases can be explained by confounding (high morbidity from cardiovascular and non-cardiovascular causes)Although incidence of coded chronic kidney disease was high (even higher incidence with eGFR), there is genetic evidence suggesting that high blood pressure is an early manifestation of underlying kidney damage.Our study included 4 million patient-years of follow-up and patients with a wide range of mean systolic blood pressures and predicted risk. The CPRD is broadly representative of the UK populationRestricting the cohort to patients with two or more blood pressure readings resulted in bias towards the inclusion of women, patients who consult their general practitioner more frequently, those who are likely to have higher blood pressures, are older, and have more comorbidities.There are some limitations of primary care data that are relevant to this study. For example, general practitioners might test kidney function more often in those with higher blood pressure; however, this detection bias would lead to more people with kidney damage being detected in the high blood pressure groups and would not fit the pattern dependent on predicted risk groups. Additionally, for diagnoses such as atrial fibrillation (which requires a confirmatory electrocardiogram), the code list used in this study might not be sensitive enough to capture all cases. However, QRISK2 code lists are not published and, therefore, we cannot confirm the accuracy of replication.The shallow gradients seen with increasing blood pressure in predicted risk groups are for 4 years of follow-up. In the long term and if left untreated, subclinical vessel damage due to high blood pressure could lead to poor outcomes, particularly stroke and vascular dementia; we also acknowledge that high blood pressure might be easier to treat and control in the early stages and leaving patients untreated for long periods might not be the optimal approach.QRISK2 is the best available validated tool in the UK primary care to predict cardiovascular risk . No risk tool will perfectly predict whether patients will have an outcome, particularly across all cardiovascular disease.Although our study mostly focuses on the White English population using the QRISK2 algorithm, because of the large sample size we were able to include a substantial number of people from other ethnic groups, albeit in smaller proportions. The principle of using risk prediction to further guide blood pressure treatment is applicable to any country that incorporates cardiovascular risk estimation into routine care. Other countries will choose the risk prediction tool which is most valid in their context.The outcomes chosen for this study were cardiovascular disease and target organ damage. We were interested in vascular dementia as an adverse effect of high blood pressure. Unfortunately, in most primary care records, the cause of dementia is unknown and, therefore, we broadened our definition of dementia to include diagnoses with no known cause.Finally, irrespective of the blood pressure cutoff guidelines for initiating treatment, we need to be mindful that blood pressure targets might be different. Our study did not investigate treatment targets and included patients who were prescribed and not prescribed blood pressure lowering drugs at cohort entry, because for most patients the treatment targets are not reached and evidence suggests lowering blood pressure even further seems not to be harmful.Our results show that within strata of predicted risk, the treatment cutoffs of 140/90 mm Hg and 160/100 mm Hg—ubiquitous in most countries—are not supported by the data in terms of an increase in rates of cardiovascular disease, target organ damage, and incident dementia compared with lower blood pressures.Patients and clinicians often focus on blood pressure measurements alone, and the data in this study show the importance of multiple risk factors driving cardiovascular risk. For individual patients making decisions about treatment, the high relative risk associated with high blood pressure should be explained in the context of their predicted absolute risk. Importantly, when medium-term predicted risk is low, there seems to be no urgency to initiate drug treatment (on the basis of low incidence rates of cardiovascular disease among this group) and the clinician might want to focus on advising patients to use non-pharmacological approaches to reduce blood pressure.At the population level, emphasis on blood pressure targets and control should not detract from focus on overall reduction of predicted risk. Most gains can be made by treating patients at highest predicted risk rather than those with high blood pressure. For blood pressures of 140/90 mm Hg and higher (including those 160/100 mm Hg and higher), although there is robust evidence of treatment benefit, the absolute gain might be small if the predicted risk is low. For blood pressures lower than 140/90 mm Hg,  if there is a real treatment benefit, then substantial gains could be made to reduce the burden of cardiovascular disease by treating a large group of patients with high predicted risk.For a wide range of blood pressures, medium-term drug treatment decisions could be effectively made on the basis of predicted absolute cardiovascular disease risk because blood pressure thresholds would add little useful information. Although blood pressure is an important part of risk prediction, a shift in the focus away from blood pressure cutoffs and targets and towards viewing blood pressure in the context of predicted risk could lead to a more effective approach for cardiovascular disease prevention.https://www.cprd.com/ and need approval of the study protocol by the Independent Scientific Advisory Committee for MHRA database research.Data were obtained from the CPRD, a research service that provides primary care and linked data for public health research. CPRD data governance and our licence to use CPRD data does not allow us to distribute or provide access to patient data to other parties. Researchers can apply for data access at DN was on the steering group for two GlaxoSmithKline funded studies investigating aspects of kidney function in children and adults in sub-Saharan Africa; holds a grant from the UK Health Foundation on improving acute cardiac care for kidney patients; and is the Renal Association's director of informatics research. LT is a member of three Medicines and Healthcare products Regulatory Agency's expert advisory groups and an unpaid member of two non-industry funded trial advisory committees. EW received personal payment for providing training on statistical methods, outside of the submitted work. All other authors declare no competing interests.


Mitochondrial rescue drugs, such as vitamin B2, are a treatment modality which can be considered in unexplained cases of cardiac arrest or impaired consciousness in which mitochondrial diseases are considered in the differential. Although case reports exist of children developing a drop in their blood pressure following administration of intravenous flavin adenine dinucleotide (FAD) sodium as vitamin B2, we present the first reported case of a child requiring cardiopulmonary resuscitation (CPR) following FAD sodium infusion for severe bradycardia and hypotension. Intravenous infusion of FAD sodium should be administered as slowly as possible, with careful monitoring of blood pressure and heart rate. Mitochondrial rescue drugs, such as vitamin B2, may be administered for suspected metabolic diseases due to cardiac arrest, impaired consciousness, or lactic acidosis with no apparent cause -4. Flavi2]: 22.6 mmHg, bicarbonate: 13.5 mmol/L, base deficit: -13.9 mmol/L, lactate: 99 mg/dL) and hyperammonemia (210 µg/dL). Because the cardiac arrest had no obvious cause, the differential diagnosis included metabolic diseases ; therefore, drugs for mitochondrial diseases, riboflavin sodium phosphate as vitamin B2 (25 mg/kg over 15 minutes twice a day), fursultiamine hydrochloride, pyridoxal phosphate hydrate, ascorbic acid, tocopherol acetate, biotin, ubidecarenone, and levocarnitine, were administered with treatment for heart failure. Her cardiac function gradually recovered without ECMO, and left ventricular ejection fraction improved from 30% to 50% on echocardiography. She was transferred back to our hospital on day seven. At the time of readmission, she had a respiratory rate of 35 breaths/min; oxygen saturation of 92% ; blood pressure of 115/55 mmHg; heart rate of 130 beats per minute (bpm) ; and axillary temperature of 36.5°C. The treatment at the previous hospital was continued, except that FAD sodium was substituted for vitamin B2, because riboflavin was not adopted by our hospital. FAD sodium, 25 mg/kg, was scheduled to be administered over one hour twice a day.The patient, a seven-month-old girl weighing 3.8 kg, had undergone pulmonary artery banding for atrioventricular septal defect and also had other congenital malformations such as hip dislocation and imperforate anus. She was hospitalized during the initial visit with a fever and cough and primary diagnosis of acute bronchitis. Five hours after admission, she suffered sudden cardiac arrest and CPR was performed. Although her heart resumed beating unassisted after one hour of CPR, her cardiac function was markedly impaired (left ventricular ejection fraction of 30% on echocardiography). Therefore, she was transferred to a facility that could immediately provide extracorporeal membrane oxygenation (ECMO). After return of spontaneous circulation, arterial blood gas analysis revealed a metabolic acidosis , her systolic blood pressure gradually dropped to approximately 60 mmHg. We decided to administer a fluid challenge to assess fluid responsiveness. Immediately after slow bolus intravenous infusion of the remaining 30 mg FAD sodium, her systolic blood pressure dropped to approximately 30 mmHg with severe sinus bradycardia (50 bpm). CPR was performed for 30 seconds due to severe bradycardia, and her blood pressure and heart rate improved immediately. The drug information for FAD sodium did not refer to hypotension, bradycardia, or cardiac arrest as adverse reactions; because her blood pressure and heart rate remained unchanged during the administration of riboflavin sodium phosphate, we assumed that the administration of FAD sodium was not attributed to bradycardia and hypotension. FAD sodium was intravenously infused again as per schedule via a slow bolus. Then, transient bradycardia (from 120 bpm to 70 bpm) and hypotension (from 112/67 mmHg to 60/27 mmHg) recurred, which improved within two minutes of discontinuation of FAD sodium infusion. We discontinued FAD sodium as the FAD sodium reproducibly affected the blood pressure and heart rate. All remaining metabolic drugs were discontinued on day 14 because the screening results were negative for metabolic disease and her clinical course had improved. Her general condition remained stable after the discontinuation of metabolic drugs.FAD sodium, formed by the binding of a riboflavin moiety vitamin B2) to a phosphatase group of an adenosine diphosphate molecule , is comm to a phoHypotension during intravenous infusion of FAD sodium has been described in several children, but as far as we know, no other children have required CPR as a result ,6. AnimaIntravenous infusion of FAD sodium should be administered as slowly as possible, with careful monitoring of blood pressure and heart rate. FAD sodium is used as a mitochondrial rescue drug. It can cause bradycardia and hypotension to the point that CPR is required. Therefore, intravenous infusion of FAD sodium should be administered as slowly as possible, with careful monitoring of blood pressure and heart rate.


FAZc was significantly reduced in greater KDIGO prognosis risk categories . VD and FAZc presented the best diagnostic performance in ROCs. In conclusion, OCTA parameters, such as VD and FAZc, are able to detect different GFR, ACR, and KDIGO categories in T1DM patients and controls in a non-invasive, objective quantitative way. FAZc is able to discriminate within T1DM patients those with greater DKD categories and greater risk of DKD progression.The purpose of this study is to investigate potential associations between optical coherence tomography angiography (OCTA) parameters and diabetic kidney disease (DKD) categories in type 1 diabetes mellitus (T1DM) patients and controls. A complete ocular and systemic examination, including OCTA imaging tests and bloods, was performed. OCTA parameters included vessel density (VD), perfusion density (PD), foveal avascular zone area (FAZa), perimeter (FAZp) and circularity (FAZc) in the superficial vascular plexus, and DKD categories were defined according to glomerular filtration rate (GFR), albumin-creatinine ratio (ACR) and KDIGO prognosis risk classifications. A total of 425 individuals (1 eye/1 patient) were included. Reduced VD and FAZc were associated with greater categories of GFR ( Diabetes mellitus (DM) is a systemic disease which affects the microvasculature of multiple organs leading to diabetic retinopathy (DR), nephropathy and neuropathy, with high impact on the quality of life of diabetic patients . DR is cOptical coherence tomography angiography (OCTA)  is a newThe aim of this report is to specifically evaluate the association between OCTA parameters and DKD stages in different classifications in a large cohort of T1DM patients and controls. The clinical relevance of establishing these associations relies on the potential ability of OCTA to estimate kidney damage stages with a non-invasive eye imaging technique. Thus, it may allow clinicians to classify patients according to their risk and prognosis and act consequently, concentrating health resources in those patients at higher risk of developing microvascular complications.ClinicalTrials.gov, accessed on 6 February 2018, NCT03422965). Written informed consent was obtained for all participants.Cross-sectional, exploratory study in a large cohort of T1DM patients with prospective collection of OCTA images and ocular and systemic clinical data. The study protocol has been described elsewhere . This prT1DM patients undergoing routine follow-up visits at the Diabetes Unit were invited to participate and referred to the Ophthalmology department for a comprehensive ocular examination. Healthy controls were also recruited from social media campaigns supported by the Communications department of the Hospital. Exclusion criteria included ocular media opacities, ocular comorbidities  and inability to perform complete ocular examinations or provide written informed consent. For this specific report, an additional inclusion criteria was the existence of kidney function tests performed within 1 month of the ocular examination.The complete details of the ocular examination and systemic status assessment of diabetes has been described elsewhere . Ocular −1), perfusion density  and foveal avascular zone (FAZ) parameters: area , perimeter  and circularity  . OCTA quantifications were performed by the device built-in commercial software  in the superficial capillary plexus (SCP). Measured variables included: vessel density  . OCTA im2 respectively). On the other hand, ACR was classified as: A1, A2 and A3 . Finally, according to GFR and albuminuria categories, CKD risk probability was assigned under KDIGO Prognosis of CKD classification: low risk (G1 or G2 and A1), moderate risk , high risk  and very high risk .To assess diabetic renal damage, kidney function tests were performed and glomerular filtration rate (GFR) estimated with CKD-EPI  and urinp-values from the generalized estimating equation (GEE) adjusted for age, gender, signal strength index (SSI), axial length, duration of DM disease and grade of DR were used. Bonferroni correction was used in multiple comparisons between group means. In nonparametric variables group mean comparisons, The Kruskal-Wallis test was used. To study the correlation between two continuous variables, the Spearman test was used. Also, receiver operating curves (ROC) were constructed to evaluate the area under the curve (AUC) of OCTA parameters. For all the tests, p-value < 0.05 was considered statistically significant. The statistical package R Studio (2.5)  was used for the statistical analyses.Descriptive analysis was performed using frequencies and percentages were for qualitative variables and mean, standard deviation (SD), median and quartiles for quantitative variables. The Kolmogorov-Smirnov test was used to assess normality of distributions. For qualitative variables, the chi-squared test was used. For quantitative parametric variables group mean comparison, A consolidated standard of reporting trials (CONSORT)-style flow diagram describing included and excluded study eyes is presented in n = 71) or lack of kidney function tests (n = 53). To avoid risk of bilaterality bias, only one eye per patient was randomly selected, and in cases with asymmetric DR stage, the eye with higher DR grade was selected. A total number of 425 individuals, 363 T1DM patients and 62 healthy controls were finally included for statistical analysis. OCTA images with artifacts (n = 36), low quality  or incorrect FAZ (n = 28) were excluded from analysis. Demographics, baseline characteristics including general characteristics, diabetes-related characteristics, such as DM duration, macrovascular complications, DR stage, systemic treatments and baseline bloods, of the study cohort disclosed by KDIGO prognosis categories are presented in A total number of 478 T1DM patients and 115 healthy controls underwent a complete ocular examination during the predetermined timeframe. Exclusion criteria were applied, and eyes were excluded due to ocular comorbidities (n ≤ 3) were excluded from analysis .Quantifications of OCTA parameters were analyzed, and intergroup differences were calculated according to GFR, ACR and KDIGO prognosis categories, as summarized in n = 263), G2 (n = 74) and G3a/G3b (n = 3). A significantly lower VD was observed in G1 and G2 categories compared to controls , overall as well as in 2 × 2 comparisons (p < 0.05 both). Similarly, lower FAZc was observed in G1 and G2 compared to controls , with significant differences between controls and G2 (p < 0.05). No differences were observed in PD, FAZa or FAZp.Mean GFR CKD-EPI calculated for T1DM cases was 100.68 ± 19.09 mL/min (mean ± SD) and 95.7 ± 22.98 mL/min for healthy controls. GFR categories included G1 (n = 318), A2 (n = 20) and A3 (n = 2). Significant differences were observed between A1 and A2 categories and controls for VD , being both 2 × 2 comparisons significant (p < 0.05). Also, FAZc was significantly reduced in A1 and A2 categories compared to controls , with significant differences in 2 × 2 comparisons for A2 vs. controls (p < 0.05) and also for A2 vs. A1 (p < 0.05). A trend was observed for PD that did not reach significance level (p = 0.08), and no differences were observed in FAZa or FAZp.Mean ACR was 9.96 ± 9.2 mg/g (mean ± SD) for T1DM and 9.5 ± 15.5 mg/g for healthy controls. ACR categories included A1 (n = 317) or “Moderate” risk . Three cases were found with greater stages  and were excluded from this analysis.T1DM patients were stratified according to the KDIGO 2012 CKD prognosis classification, based on the combination of the previous GFR and ACR categories. Two groups were identified as “Low” risk , with significant differences in both 2 × 2 comparisons. FAZc also showed significant differences and lower values were observed in “Low” and “Moderate” risk categories compared to controls . In 2 × 2 comparisons, significant differences were observed in FAZc between “Moderate” risk and controls (p < 0.05) and also “Low” risk patients (p < 0.05). PD showed a trend that was not significant (p = 0.06) and no differences were observed in FAZa or FAZp.VD was significantly reduced in patients with “Low” and “Moderate” risk categories compared to controls , and no associations were observed for any other parameter, DR grade or DM duration groups. For ACR, a non-significant negative trend was observed for FAZc in T1DM with >15 years of DM duration (p = 0.08). No correlations were observed between ACR and any OCTA parameters, DM duration or DR grade.Correlation tests (Spearman’s rank correlation coefficient) were performed between OCTA parameters and GFR and ACR, overall and stratified by DM disease duration  and DR grades . For GFR, a non-significant positive trend was observed for FAZa in proliferative DR (Receiver operating curve (ROC) analysis were constructed to evaluate the diagnostic utility of OCTA parameters to detect patients with “low” and “moderate” risk of CKD according to KDIGO categories in non-DR and DR patients, presented in This report demonstrates the ability of specific OCTA parameters to identify different DKD categories in a non-invasive, objective quantitative way, using retinal images and blood test data from a large cohort of T1DM patients and controls collected prospectively. We describe that VD and FAZc parameters are able to detect different GFR, ACR and KDIGO categories in T1DM patients and controls, and we report that FAZc is able to discriminate within T1DM patients those with greater categories of DKD, and more importantly, those at greater risk of DKD progression. These findings highlight the potential of OCTA as a non-invasive tool to identify patients at risk of DKD progression in both specialized units and the community.n = 10, 70% Caucasian, T2DM) did not found any association between VD and GFR, and only described an association with peripheral non-perfusion areas in wide field OCTA montages. Finally, some of these series present variable control of DM disease, as reflected in the wide range of HbA1c levels (6.7 to 9.4%) described. For all these reasons, the results reported in this study add to the existing evidence that VD and DKD appear to be associated in T1DM individuals.Since the advent of OCTA , severalOne of the most clinically relevant findings of this study is the identification of FAZc as a potential indirect biomarker of DKD progression. The status of the FAZ and its different parameters, such as area, perimeter or circularity, has been previously described as a marker associated with DR stage ,15,18, aROC curves have been constructed with the aim to evaluate the diagnostic potential of OCTA parameters for each category and DKD classification, as well as to investigate this performance separately in non-DR cases and cases with DR. In both subgroups, the greatest AUC values were observed for VD and FAZc to identify cases with greater categories of ACR (A2) and KDIGO (Moderate risk) classifications. These results suggest that OCTA may be useful to detect subgroups of patients with kidney disease, or even more importantly, identify patients at risk of DKD progression in a non-invasive way. The implications of this finding are relevant as a blood test is currently required to assess this risk. In this area of research, recent studies have highlighted the potential of artificial intelligence (AI) algorithms to identify CKD categories from retinal photographs using existing datasets from DR screening programs ,42. HoweThis study has a number of strengths and limitations. The large study cohort, recruited prospectively with collection of high-quality ocular and systemic data, the strict exclusion criteria applied to cases and OCTA images, the selection of 1 eye per patient to avoid risk of bilaterality bias and the presence of a control group are the main strengths of this study. Limitations include the use of a commercial software that allows the evaluation of only the SCP and not the DCP and the lack of T1DM patients with advanced categories of CKD.In conclusion, this study demonstrates that specific OCTA parameters, such as VD and FAZc, are able to differentiate distinct DKD categories in a non-invasive, objective and quantitative way in T1DM patients and controls. Moreover, we specifically describe that FAZc could considered a biomarker of DKD progression in T1DM individuals, being able to identify patients at greater risk according to the KDIGO prognosis classification. If these findings are confirmed in future studies, OCTA may play a relevant role in the management of DKD in different scenarios, such as highly specialized Diabetes units and, potentially, deployed in a community setting, as a non-invasive helpful tool to identify patients with greater categories of DKD or higher risk of DKD progression.