Endothelial dysfunction in the kidney transplant population: Current evidence and management strategies

Abstract

The endothelium modulates vascular homeostasis owing to a variety of vasoconstrictors and vasodilators. Endothelial dysfunction (ED), characterized by impaired vasodilation, inflammation, and thrombosis, triggers future cardiovascular (CV) diseases. Chronic kidney disease, a state of chronic inflammation caused by oxidative stress, metabolic abnormalities, infection, and uremic toxins damages the endothelium. ED is also associated with a decline in estimated glomerular filtration rate. After kidney transplantation, endothelial functions undergo immediate but partial restoration, promising graft longevity and enhanced CV health. However, the anticipated CV outcomes do not happen due to various transplant-related and unrelated risk factors for ED, culminating in poor CV health and graft survival. ED in kidney transplant recipients is an under-recognized and poorly studied entity. CV diseases are the leading cause of death among kidney transplant candidates with functioning grafts. ED contributes to the pathogenesis of many of the CV diseases. Various biomarkers and vasoreactivity tests are available to study endothelial functions. With an increasing number of transplants happening every year, and improved graft rejection rates due to the availability of effective immunosuppressants, the focus has now shifted to endothelial protection for the prevention, early recognition, and treatment of CV diseases.

Key Words: Endothelial dysfunction; Endothelium; Cardiovascular disease; Kidney transplantation; Chronic kidney disease; Nitric oxide; Flow-mediated dilatation; Nitro-glycerine-mediated dilatation; Biomarkers

Core Tip: Endothelium promotes vascular homeostasis by regulating vascular permeability, leucocyte diapedesis, hemostatic response, and vascular tone. Endothelial dysfunction (ED) plays a key role in the genesis of cardiovascular (CV) and kidney outcomes seen in the chronic kidney disease and kidney transplant population. There is increased CV morbidity and mortality imparted due to ED in transplant recipients. Among various biomarkers and diagnostic tests available to detect endothelial function, flow-mediated dilation is a widely recognized and reproducible test available in clinical settings. Though overlooked in kidney transplant settings, promoting CV and graft health by monitoring endothelial functions and mitigating risk factors of ED can improve overall outcomes.

INTRODUCTION

The endothelium, which forms the inner layer of the blood vessels, actively regulates vascular functions by sensing and responding to mechanical and biochemical signals. This vasoregulation happens through modulating vascular tone, mediating cellular adhesion, influencing the migration of inflammatory cells, and providing resistance to the action of pro-thrombotic and pro-inflammatory mediators. Under a homeostatic state, endothelial cells release nitric oxide (NO) under the influence of endothelial NO synthase (eNOS)[1]. NO is one of the major mediators of endothelial functions that prevents platelet aggregation and causes vasodilation by modulating the tone of vascular smooth muscle cells. In addition to NO, fibrinolytic and antithrombotic properties of the endothelium are also mediated through other mediators such as tissue plasminogen activator, antithrombin III, and prostacyclin[2]. Bone marrow-derived endothelial progenitor cells are responsible for the renewal of endothelial cells during repair.

Endothelial dysfunction (ED) is marked by a decrease in NO production/availability and an inflammatory endothelial cell phenotype (activated endothelial cells). There is poor expression of eNOS following damage to the endothelial glycocalyx, a gel-like layer of glycoproteins and extracellular matrix components involved in trans-endothelial transport at the blood-endothelial interface[3]. Additionally, there is eNOS uncoupling, leading to the generation of reactive oxygen species (ROS) instead of NO[4]. Thus, there is decreased NO availability and production. Activation of endothelial cells (novel immune cells) by various inflammatory signals results in the upregulation of cellular adhesion molecules, including e-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell-adhesion molecule-1 (VCAM-1), which promote inflammation. Subsequently there is leukocyte diapedesis in conjunction with increased endothelial permeability[2]. Activated endothelial cells behave like macrophages, secreting cytokines. The disturbed and inflamed endothelium favors permeation and trapping of lipoprotein particles beneath the endothelial cell layer, which can subsequently get oxidized[2]. The deposited lipoproteins are internalized by macrophages and smooth muscle cells, resulting in the formation of foam cells and the accumulation of fatty streaks. Altered efferocytosis (removal of apoptotic endothelial cells) leads to vascular stiffening[5]. Some endothelial cells may undergo endo-mesenchymal transition, facilitating deposition of the extracellular matrix, thereby further aggravating vascular stiffness[6]. Arterial constrictions due to lipid accumulation in conjunction with poor vasodilation increases regions of turbulent or oscillatory blood flow, which further exacerbates endothelial cell activation and denudation. Lipid-rich plaques with a thin fibrous cap in these areas are at high risk for rupture, causing thrombotic occlusions. This risk is exacerbated by the expression of pro-coagulant tissue factor, anti-fibrinolytic plasminogen activator inhibitor-1 (PAI-1), and von Willebrand factor (vWF)[2,7]. Thus, ED is a forerunner for atherosclerosis and thrombosis.

In this review, we examine cardiovascular (CV) diseases prevalent in chronic kidney disease (CKD) and transplant settings, focusing on their pathogenesis as it relates to ED. We emphasize the inadequacies in the prevention, diagnosis, and management of ED in real-world scenarios. By correlating the pathogenesis of ED with its diagnostic and therapeutic approaches, we aim to provide valuable insights for clinicians. We hope that this review will inspire researchers to undertake collaborative studies with clinicians, thereby advancing our understanding of ED in the context of kidney transplantation.

CV diseases due to ED in CKD

The decline in glomerular filtration rate in CKD leads to a fast-tracked CV aging trajectory. Patients with advanced CKD (stage 4 and 5) have the highest risk of dying from CV diseases. Almost 50% of all deaths in advanced CKD are due to CV diseases[8]. In addition to coronary artery diseases, heart failure (with both reduced and preserved ejection fraction), arrhythmias, valvular abnormalities, peripheral arterial disease, and sudden cardiac death are also commonly observed in the CKD population[8]. ED contributes significantly to increased CV risk in patients with CKD. CKD is characterized by persistent low-grade inflammation due to oxidative stress, cardio-metabolic risk factors, infections, uremic toxins, acidosis, and mineral-bone alterations, all of which damage the endothelium[9,10]. Concrete evidence suggests the role of inflammation in causing CV diseases through the activation of endothelial cells in patients with CKD[11]. Levels of pro-inflammatory cytokines such as interleukins (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), and high-sensitivity C-reactive protein (hs-CRP) positively correlate with increased CV risk in CKD[12]. Anti-inflammatory therapy with canakinumab (targets IL-1β) has shown an 18% reduction in the primary endpoint (non-fatal myocardial infarction, non-fatal stroke, or CV death) in patients with moderate CKD[13]. Oxidative stress and uremic toxins of CKD state induce pro-inflammatory signals and cause post-translational modification (oxidation and carbonylation) of both low-density lipoprotein (LDL) and high-density lipoprotein (HDL), which are CV risk factors[12]. Thus, alterations in lipoprotein particle profiles in CKD exacerbate the atherogenicity of LDL, and transform HDL from a protective to a potentially harmful entity. In addition to the above risk factors, metabolic acidosis, decreased levels of klotho (an anti-vascular aging protein), vascular calcification induced by phosphate alterations, and altered gut microbiome contribute to ED in CKD[12]. Dialysis modalities are also implicated in causing CV diseases like pulmonary hypertension through ED[14,15]. Hemodialysis (HD) patients have higher endothelial microparticles and pro-inflammatory monocytes [cluster of differentiation (CD) 14 + CD16 + monocytes] than patients on peritoneal dialysis (PD)[16].

In the clinical setting, a cohort study from India by Prasad et al[17] analyzing practice patterns in mild to moderate CKD, showed that renin-angiotensin-aldosterone system (RAAS) blockers were prescribed to 47.9% of CKD patients overall and 58.8% of those with proteinuria, while only 40.4% were on statins. Both the drug classes are protective to the endothelium. This highlights a missed opportunity in CKD to safeguard endothelial function due to non-compliance with guideline recommendations. Statins lower plasma cholesterol levels and decrease the endothelial trapping of modified lipoproteins. Statins also exhibit “pleiotropic” vascular benefits, including enhancing endothelial function by increasing NO bioavailability by upregulating eNOS, endothelial repair, reducing oxidative stress, and inhibiting inflammation. Statins also decrease the levels of endothelin-1, a potent vasoconstrictor[18]. In adults with dialysis-dependent CKD (unlike CKD patients not initiated on dialysis), the Kidney Disease: Improving Global Outcomes (KDIGO) recommend against starting statins or statin/ezetimibe combinations[19]. Although CV risk is elevated in this subgroup of CKD, the effectiveness of statins is uncertain due to significant competing mortality risks. RAAS activation promotes vascular stiffness by promoting pro-fibrotic and inflammatory pathways. RAAS activation causes smooth muscle cell proliferation, extracellular matrix deposition, and transforming growth factor β (TGF-β) secretion (pro-fibrotic)[20]. Despite the benefits, many patients with CKD are discontinued from RAAS inhibitors because of adverse events[21]. There is less inclusion of CKD patients in CV clinical trials despite calls for better representation[22]. Thus, in CKD, there is an unchecked and progressive endothelial damage. This culminates in increased CV risk as the patient prepares for a kidney transplant.

CV diseases after kidney transplant

Kidney transplant candidates have a notable prevalence of both pre-existing (risk factors acquired during CKD state) and de novo transplant-related CV risk factors, which contribute to adverse CV outcomes[23]. The traditional CV risk factors in the post-transplant period include hypertension (40%-90%), diabetes (24%-42%), dyslipidemia (50%), smoking (25%), and obesity. The non-traditional CV risk factors specific to kidney transplant settings include adverse effects of immunosuppressive medications, persistent secondary hyperparathyroidism after transplant, anemia, chronic inflammation, and graft dysfunction[23,24]. The spectrum of CV diseases in kidney transplant settings does not differ greatly compared to the CKD population, and include coronary artery disease, valvopathy, heart failure, stroke, cardiac arrhythmias, pulmonary artery hypertension, and peripheral arterial disease.

Despite comorbidities, patients with CKD after transplantation exhibit improved CV health compared to those who are waitlisted for transplant[25]. Kidney transplantation significantly improves maximal oxygen consumption (from 20.7 to 22.5 mL/kg/minute, P < 0.001) compared to those who remain on dialysis (declines from 18.9 to 17.7 mL/kg/minute, P < 0.001) at 1 year of follow-up[26]. Similarly, improvements were seen with ejection fraction in a kidney transplant group (improved from 60.1% to 63.2% P = 0.02) compared to a waitlisted group (61.4% to 59.3%)[26]. However, not all risk factors that are acquired in the pre-transplant period are mitigated. The leading cause of death with a functioning graft in the long-term remains due to CV causes[27]. There is a high incidence of myocardial infarction, heart failure, and stroke in the 1^st year after kidney transplant [cumulative incidence in the 1^st year is 2.6%, 95% confidence interval (CI): 1.9-3.2], and the risk continues to increase in the subsequent post-transplant years (cumulative incidence between the years 2-5 is 8.3; 95%CI: 7.1-9.5)[28].

There is a significant spillover of CV risk factors acquired from the pre-transplant period. Among 62706 kidney transplant recipients, 6.4% had atrial fibrillation before transplant. Over a mean follow-up of 4.9 years, those with atrial fibrillation experienced higher mortality (40.6% vs 24.9%), greater all-cause graft failure (46.8% vs 36.4%), and higher rates of ischemic stroke (2.8% vs 1.6%) after transplantation[29]. In a cohort of 1063 kidney transplant recipients, left ventricular hypertrophy [hazard ratio (HR) = 1.58; P = 0.022] and increased relative wall thickness (HR = 1.44; P = 0.041) before transplant independently predicted CV events after transplant. Persistent brachiocephalic fistula is also a risk factor for pulmonary arterial hypertension. In addition to the spillover of CV diseases, de novo CV diseases after transplantation do occur. The cumulative incidence of de novo congestive heart failure after kidney transplantation is 10.2% at 12 months and 18.3% at 36 months after transplant[30].

ED as a risk factor for increased CV risk

A recent review article by Cardinal et al[31] briefly addresses the immunopathogenesis of endothelial injury following kidney transplantation, with a primary focus on renal endothelium. ED extends beyond the kidneys and is a precursor to various CV diseases. Although direct evidence linking ED to different CV diseases in kidney transplant settings is sparse due to a limited number of studies, recent data suggest that a significant proportion of CV diseases in these settings may be attributable to ED. Also, as pointed out by Cardinal et al[31], ED damages the graft endothelium and causes graft failure.

The cumulative incidence of post-transplant myocardial infarction (PTMI) was 4.3% at 6 months, 5.6% at 12 months, and 11.1% at 36 months[32]. The role of ED in atherogenesis has already been explained. The high incidence of PTMI early post-transplant may be attributed to the high levels of inflammatory markers such as vWF, PAI-I, and homocysteine levels[33]. A hospital-based registry from the United States[34] showed notable increases in admissions among transplant recipients between 2005 and 2011 for congestive heart failure and cardiac arrhythmias (P = 0.01), which accounted for 16% to 17% of all hospital admissions. Coronary ED leads to heart failure with preserved ejection fraction[35]. Pre-emptive transplantation may reduce the risk of heart failure, and there is a lesser risk of coronary ED by avoiding HD[36]. The incidence of stroke after kidney transplantation varies from 1.1% to 8% and ischemic stroke is the most common variant. The risk of rupture of intracranial aneurysms may theoretically be increased in patients with autosomal dominant polycystic kidney disease (ADPKD). ADPKD is notably associated with RAAS activation and ED[37]. However, data show favorable CV outcomes in patients with ADPKD, including stroke[38]. In Cox regression analysis, modeling kidney transplant as a time-dependent covariate showed an adjusted HR (aHR) of 0.77 (95%CI: 0.67-0.89) for intracranial hemorrhage in ADPKD patients during the post-transplant period compared to the pre-transplant period[39]. The 3-year cumulative incidence of de novo peripheral arterial disease after kidney transplant is 20% among CKD patients with diabetes[40]. Ankle brachial index before transplant was an independent predictor of graft failure [odds ratio (OR) = 2.77; 95%CI: 1.68-4.58; P < 0.001] and secondary CV endpoints, namely myocardial infarction; cerebrovascular accident; and limb ischemia, gangrene, or amputation (HR = 1.39; 95%CI: 0.97-1.99; P = 0.076), and death (HR = 1.84; 95%CI: 1.26-2.68; P = 0.002)[41]. Important regulators of pulmonary circulation include endothelin-1 and NO, which are therapeutic targets in treating pulmonary hypertension[42]. One etiopathogenesis of pulmonary arterial hypertension is due to ED caused by arterio-venous fistula created for dialysis access[43]. Fistula ligation may be a reasonable option in patients with pulmonary hypertension after transplant.

RISK FACTORS FOR ED IN THE KIDNEY TRANSPLANT POPULATION AND CLINICAL IMPLICATIONS

Traditional risk factors

Obesity: The protective impact that obesity might have on patients undergoing dialysis disappears following a transplant. In obesity, ED is primarily driven by adipose tissue inflammation (increased IL-6, TNF-α), decreased expression of eNOS, insulin resistance, and oxidized LDL (oxLDL)[44]. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of eNOS, is elevated in obesity[44]. Weight gain happens in the 1^st year after transplant, especially in children[45]. Steroid minimization is associated with lesser weight gain[46]. Obesity in transplant candidates is also a risk factor for diabetes mellitus[47], and sleeve gastrectomy in obese kidney transplant patients improved blood pressure and glucose control[48]. Pre-transplant obesity is a risk factor for delayed graft function[49]. Obstructive sleep apnea (OSA) associated with obesity may re-emerge long-term in transplant recipients, even if symptoms of OSA initially improved after the transplant[50]. OSA decreases NO production due to repeated episodes of hypoxia, and is a risk factor for pulmonary arterial hypertension[51,52].

Clinical vignette: The role of novel anti-obesity and anti-diabetic agents, particularly glucagon-like peptide-1 (GLP-1) analogs, in modulating the effects of ADMA represents a promising area of research.

Smoking: Smoking in kidney transplant candidates is associated with high rates of coronary artery disease[53], graft loss, and non-CV mortality[54]. Almost 50% of the kidney transplant population in a cohort study were current or former smokers. Smoking promotes ED by the generation of ROS, increased oxLDL, inactivation of eNOS, and increased expression of cell-adhesion molecules. Smoking cessation has been shown to improve endothelial functions. Among 1504 smokers, flow-mediated dilatation (FMD) (described in detail below) improved by 1% (P = 0.005) after 1 year in those who quit smoking, while remaining unchanged in those who continued (P = 0.643)[55].

Clinical vignette: Increased public awareness of the health risks associated with tobacco has driven many to use electronic cigarettes (e-cigarettes) as a potentially less harmful alternative to smoking. On the contrary, e-cigarettes also cause ED. E-cigarette users and smokers showed lower FMD compared to non-users[56]. They demonstrated higher concentrations of receptors for advanced glycation end products (RAGE) than smokers[56]. RAGE ligands amplify pro-inflammatory responses in the endothelial cells.

Dyslipidemia: In the initial year after kidney transplantation, dyslipidemia is seen in 60%-88% of both adult and pediatric patients, surpassing the prevalence seen in the general population[57]. Most of the commonly used immunosuppressives are associated with abnormal lipid metabolism, especially cyclosporine and mammalian target of rapamycin (mTOR) inhibitors[57]. Cyclosporine and corticosteroids have an additive effect on raising cholesterol, with partial improvement upon steroid withdrawal[58]. Conversely, mTOR inhibitors significantly increase both cholesterol and triglycerides in a dose-dependent manner[58]. Belatacept-based immunosuppression is associated with improved LDL levels[59]. The role of oxLDL and carbamylated LDL in atherogenesis has already been mentioned. OxLDL levels correlated with increased proteinuria and graft fibrosis in a porcine renal transplantation model[60].

Statin use was linked to lower mortality in kidney transplant recipients (aHR = 0.95; 95%CI: 0.90-0.99)[61]. A trial showed that atorvastatin was more effective than switching from cyclosporine to tacrolimus in reducing total cholesterol and LDL, with additional LDL reduction when combined with tacrolimus[62]. Atorvastatin increased FMD from 4.0% to 6.5% (P < 0.05), while tacrolimus slightly improved FMD to 5.1% (P < 0.05), but adding atorvastatin to tacrolimus had no significant effect on FMD[62]. The role of statins (both LDL lowering and pleiotropic functions) in preventing ED has already been discussed. According to the KDIGO, kidney transplant recipients are advised to receive statin therapy irrespective of their LDL concentrations. The usage of statins in kidney transplant recipients is still 69.1% only as per the 2021 United States renal data system annual report[63].

Clinical vignette: The role of proprotein convertase subtilisin/kexin type 9 antibodies on lipid abnormalities and endothelial functions is still unexplored in the transplant recipients[64].

Hypertension: Hypertension is present in 50%-80% of kidney transplant recipients[65,66]. In the post hoc analysis of folic acid for vascular outcome reduction in transplantation trial, each 20-mmHg rise in baseline systolic blood pressure correlates with a 32% elevation in subsequent CV risk (HR = 1.32; 95%CI: 1.19-1.46) and a 13% increase in the risk of mortality (HR = 1.13; 95%CI: 1.01-1.27)[67]. The chances of graft failure in poorly controlled hypertensives are twice that of controlled hypertensives[68]. Inflammation is believed to play an important role in the pathogenesis of hypertension[69]. Chronic sympathetic nervous system activation, RAAS activation, obesity, high salt intake, immunosuppressive drugs, graft dysfunction, infections, and oxidative stress promote the release of pro-inflammatory cytokines causing endothelial damage leading to hypertension and further target organ damage[69]. Donor risk factors (age, hypertensive donors, graft renal artery stenosis, donor smoking, dyslipidemia, atherosclerosis) also play an important role[70]. Drugs such as calcineurin inhibitors (CNI), which includes both cyclosporine and tacrolimus, are hypothesized to enhance the expression of angiotensin II type 1 receptors and reduce NO production, leading to renal vasoconstriction[66]. Calcium channel blockers are theoretically beneficial in patients on CNI by facilitating calcium entry into vascular smooth muscles, leading to vasodilatation[66]. Posterior reversible encephalopathy syndrome in kidney transplant recipients associated with CNI usage is attributable to ED[71]. Spironolactone, a mineralocorticoid antagonist, has been shown to improve ED in experimental rat models[72]. However, a randomized controlled trial among kidney transplant recipients failed to show the effect of spironolactone in mitigating ED[73]. Despite increased plasma aldosterone levels with spironolactone treatment, there were no significant improvements in markers of ED such as nitrite, nitrate, cyclic guanosine monophosphate, arginine, citrulline, ornithine, ADMA, symmetric dimethylarginine, NG-monomethyl-L-arginine, vWF, tPA antigen, PAI-1 antigen and markers of inflammation-hs-CRP, ICAM, VCAM, and serum amyloid protein A compared to a placebo, except for a decrease in nitrite levels observed in patients with diabetes[73]. These findings suggest that mineralocorticoid receptor antagonism alone may not effectively mitigate ED or vascular inflammation prevalent in kidney transplant patients. This warrants further research to assess its potential benefits on vascular outcomes[24].

Higher salt intake (6%) in kidney transplant candidates often contributes to poor control of hypertension[74]. Higher salt is associated with post-prandial ED measured through FMD[75]. Hence, salt intake should be restricted for kidney transplant candidates. ED in hypertensive renal transplant recipients is linked to elevated ambulatory blood pressure and heightened BP variability (BPV), irrespective of mean blood pressure levels[76]. Notably, increased BPV in 24-hour systolic and diastolic readings is associated with ED measured through FMD[77]. Thus, ambulatory blood pressure monitoring in kidney transplant individuals has a significant diagnostic utility in preventing ED-related target organ damage[76].

Clinical vignette: Promising novel approaches for hypertension include cyclic guanosine monophosphate augmentation, RAAS inhibition using aldosterone synthase inhibitors and silencing RNA, and antisense therapies targeting angiotensinogen[78]. Theoretically, they all improve the production of NO and can have endothelial-protective effects; however, studies on transplant recipients are lacking.

Diabetes mellitus: Other than those who have pre-existing diabetes mellitus before transplantation, new-onset diabetes after kidney transplant (NODAT) develops in 10%-40% of kidney transplant recipients[79]. Though kidney transplantation improves microvascular outcomes in type I diabetic patients with simultaneous pancreas-kidney transplant recipients[80], most type II diabetes patients after kidney transplantation suffer from CV complications arising due to ED[81]. Hyperglycemia post-kidney transplant is significantly associated with an increased risk of major cardiac (HR = 1.113), vascular (HR = 1.168), and cerebrovascular events (HR = 1.156)[82]. Oxidative stress, mitochondrial fusion abnormalities, and inflammation play an important role in hyperglycemia-induced ED[83,84]. Some of the transplant-related immunomodulatory risk factors that cause both NODAT and ED include immunosuppressive drugs (corticosteroids, CNI, and mTOR inhibitors) and infections with hepatitis C virus and cytomegalovirus[79]. Sitagliptin, a dipeptidyl peptidase-4 inhibitor, showed no improved vasoreactivity as measured by laser doppler flowmetry or meaningful changes in the levels of inflammatory markers such as CRP, VCAM-1, adiponectin, and soluble TNF receptor-1[85]. However, metformin improves endothelial functions in non-transplanted subjects with type II diabetes[86]. A systematic review is underway to assess the effects of metformin on graft and patient survival in transplant recipients with type II diabetes mellitus[87].

Clinical vignette: Recommended measures to improve endothelial functions include reduced steroid/steroid-free immunosuppression in patients with low immunological risk, early post-operative basal insulin therapy for hyperglycemia for NODAT prevention, usage of newer anti-diabetic drugs with proven CV benefits like sodium glucose-2 inhibitors (SGLT-2 inhibitors), and GLP-1 analogs[88-91]. SGLT-2 inhibitors may improve endothelial functions by reducing oxidative stress, modulating adhesion molecules, and decreasing pro-inflammatory cytokines[92].

Non-traditional risk factors

Role of inflammation, toxins, infections, and oxidative stress: CKD is associated with retention of uremic solutes, inflammation, and oxidative stress. Factors causing inflammation in CKD include altered lipoprotein profiles, the gut microbiome, biomarkers of accelerated aging, oxidative stress, RAAS, and calcium-phosphate homeostasis. CKD and inflammation are interconnected in a bidirectional loop, where both exacerbate each other. The inflammatory state induces post-translational modifications of proteins in vascular biology, including the formation of glycated albumin, oxLDL, carbamylated sortilins, and AGEs[12,93]. They cause vascular calcification[94]. The inflammatory state leads to platelet activation, resulting in adherence to the endothelium and subsequent pathological thrombosis, which increases the risk of bleeding[95]. In CKD, dysbiosis of the gut microbiome results in the accumulation of bacterial-derived toxins such as endotoxins, indoxyl sulfate, p-cresyl sulfate, trimethylamine N-oxide, and phenylacetylglutamine[11]. These toxins can cause ED. A distinct population of monocytes, human leukocyte antigen (HLA)-DRhiIMs isolated from blood, produce higher amounts of TNF-α and IL-1β and is elevated in CKD[96].

Though kidney transplantation is associated with improved clinical outcomes, the inflammatory milieu of CKD continues to exist even after kidney transplant. Uremic toxins like ADMA (their role has been previously described) have been shown to persist in kidney transplant settings, and correlate with excess mortality and graft failure rates[97,98]. Hyperuricemia, hyperglycemia, hypertension, obesity, and dyslipidemia, which are common in the post-transplant state, cause ED[99]. Further, infections by immunomodulatory viruses such as cytomegalovirus, which are common in kidney transplant recipients, are known to cause expansion of pro-inflammatory and pro-thrombotic cell lines[100,101]. Coronavirus disease 2019 (COVID-19) is also known to cause similar immuno-thrombotic states in kidney transplant recipients[95]. Sepsis-induced acute kidney injury affects renal microvascular endothelial responses[102]. The gut microbiome in kidney transplant recipients is altered due to immunosuppressants and graft function[103].

Clinical vignette: Anti-inflammatory therapies like canakinumab and colchicine targeting atherosclerosis have never been tried in transplant settings. As transplantation is a risk factor for malignancy, the usage of immune checkpoint inhibitors for treating cancers can cause ED through the production of cytokines, and CV risk prevention is advised[104].

Uric acid: Purine metabolism through xanthine oxidase (XO) generates ROS, in addition to the production of uric acid[105]. ROS decreases NO availability through eNOS uncoupling. Uric acid, by itself, causes ED by entering the endothelial cells through uric acid transporters[105]. In transplant settings, hyperuricemia is one of the manifestations of CV toxicity[106]. In a study of 307 kidney transplant recipients, 47% had hyperuricemia at 6 months after transplant[107]. Over 4.3 years, hyperuricemia was associated with increased rates of new CV events and allograft dysfunction[107]. Serum uric acid levels negatively correlated with FMD in transplant settings[108]. Hyperuricemia was associated with higher anti-hypertensive medication use[109]. Allopurinol (an XO inhibitor) has been shown to improve FMD and reduce left ventricular mass in CKD patients, though no such studies have evaluated the same in kidney transplant recipients[110]. As mentioned earlier, colchicine (used in gout) has been recently added to the current standard of CV care[111].

Clinical vignette: The mechanism behind elevated blood pressure in hyperuricemic individuals involves both oxidative stress and intracellular urate activity, suggesting that anti-hyperuricemic drugs could be effective in controlling blood pressure[112].

Immunosuppressive drugs: Cyclosporine induces the release of endothelial microparticles, markers of endothelial activation[113]. Endothelial microparticles activate components of alternate complement pathways, leading to complement-mediated CV inflammation[114]. CNIs, both tacrolimus and cyclosporine, induce TGF-β production, which promotes endothelial-to-mesenchymal transition[115]. A small study of 31 renal transplant patients on cyclosporine and tacrolimus showed that the FMD in the cyclosporine group was significantly lower compared to healthy controls (6.3% vs 11.9%; P = 0.024). In contrast, no difference existed between the tacrolimus group and controls (8.8% vs 11.9%)[116]. CNI cause thrombotic microangiopathy (TMA) and chronic allograft nephropathy through ED.

Mycophenolate mofetil (MMF) has been shown to inhibit T-lymphocyte adhesion and penetration, indicating its potential as a therapeutic agent for mitigating ED in transplant recipients[117].

The “mTOR inhibitors” refer to medications that target the mTOR, a critical regulatory protein kinase involved in various cellular processes such as cell growth, proliferation, and metabolism. Sirolimus and everolimus are drugs used in both transplant settings and cardiology. mTOR inhibitors are associated with hyperuricemia, dyslipidemia, rejection, and proteinuria, which can cause ED[118]. In a prospective parallel-group study with a 7-month follow-up, sirolimus combined with MMF demonstrated superior FMD compared to MMF combined with cyclosporine[116].

Belatacept, a co-stimulation blocker of T cells used in CNI minimization strategy, has been associated with better metabolic and CV side effects than CNIs[119,120]. In the BENEFIT-EXT trial, which included elderly patients with comorbidities, the rate of serious CV events was 5.2 per 100 patient-years with cyclosporine and 4.1 per 100 patient-years with belatacept, representing a 21% relative risk reduction with belatacept[121].

Clinical vignette: Belatacept has been tried successfully as a rescue agent in de novo drug-induced TMA, which is caused due to endothelial activation[122].

Ischemia-reperfusion injury: Ischemia-reperfusion injury (IRI) is a complex process occurring when blood supply returns to tissues after a period of ischemia, leading to tissue damage and delayed graft function, especially in deceased donor kidney transplants. ED is a key component of IRI as reperfusion leads to endothelial cell death by releasing ROS[123]. There is consequent inflammation and thrombosis due to the activation of complement and coagulation pathways. Delayed graft function is associated with increased CV mortality and death-censored graft failure[124,125].

Clinical vignette: To prevent delayed graft function due to IRI strategies, include thymoglobulin administration, shorter cold ischemia time, hypothermic machine perfusion, and improving outcomes of higher kidney donor profile index organs[125,126].

Allograft dysfunction and rejection: Allograft dysfunction caused by both chronic T-cell-mediated and antibody-mediated rejection is characterized by endothelial damage[127]. Usage of CNIs causes allograft vasculopathy due to ED. Also, graft dysfunction is associated with poorly controlled hypertension with resultant downstream adverse CV outcomes[128].

HLA antigens are expressed on the endothelial surfaces[129]. Both complement and non-complement binding HLA antibodies, responsible for antibody-mediated rejection damage the endothelium[130,131]. Non-HLA antibodies like anti-angiotensin-1 receptor antibodies can also damage the endothelium[132]. Accelerated arteriosclerosis in kidney allografts is 2.9 times more common when circulating antibodies are present[133].

The therapies available to treat antibody-mediated endothelial damage are limited. Mineralocorticoid antagonists and aldosterone synthase inhibitors were unable to prevent chronic allograft injury in clinical trials[134]. Therefore, the prevention and effective management of rejection may offer prospects for long-term improvement in endothelial function.

Clinical vignette: Donor-derived cell-free DNA generated from donor-cell damage or death is an emerging non-invasive biomarker employed to study endothelial injury in the allograft[135].

Risk factors implicated due to dialysis modality: Microcirculatory dysfunction is a well-described phenomenon with dialysis, more with HD than with PD. Circulatory stress during HD can cause acute, reversible segmental myocardial hypoperfusion and regional systolic contractile dysfunction (myocardial stunning). Repeated episodes result in cumulative injury, leading to heart failure[136]. Dialysis membranes and tubing have been shown to activate complement and the factor XII-driven contact system of coagulation[137]. This activation stimulates the intrinsic coagulation pathway and the kallikrein/kinin system, contributing to CV injury through pro-coagulant and pro-inflammatory effects[137]. Endothelial glycocalyx components, including hyaluronan, syndecan-1, and hyaluronidase activity are elevated in patients with HD[138]. HD is commonly associated with increased oxidative stress[139]. Compared to PD, HD arteries exhibit reduced contractility and endothelium-dependent relaxation[140]. ED has also been demonstrated with PD. Brachial FMD was significantly lower in PD patients (2.9%) compared to controls (6.2%; P < 0.001) in a prospective study[141].

Clinical vignette: Microcirculatory protection during HD includes maintenance of BPs within an optimal range, avoiding large-volume ultra-filtration rate, daily dialysis, dialysate cooling, ischemic pre-conditioning, and combining PD with HD[142]. Hemodiafiltration, which removes middle molecular proteins, caused improvement in FMD compared to HD[143].

Native kidney diseases: Patients with nephrotic syndrome demonstrate poor FMD when their disease is active[144]. Nephrotic syndrome broadly includes minimal change disease, focal segmental glomerulosclerosis (FSGS), and membranous nephropathy. The pathophysiological processes underlying the development of atherosclerotic lesions and FSGS appear to overlap, involving shared mechanisms such as endothelial cell injury, macrophage infiltration, dyslipidemia, and hypertension[145]. Soluble urokinase-type plasminogen activator receptor, a novel biomarker released from endothelial cells and leukocytes, is associated with FSGS recurrence and the development of coronary ED[146,147]. Other markers of endothelial activation such as vWF, VCAM, syndecan-1, and e-selectin are also elevated in nephrotic syndrome[148]. FSGS recurrence after kidney transplant is also associated with the production of angiotensin-receptor-1 antibodies, which are known to damage the renal endothelial cells[149]. FSGS recurrence is estimated to occur in 30%-50% of patients with transplantation. Hence, ED can be assumed to be significant in patients who had nephrotic syndrome before transplant and those who had recurrences in their allograft.

The disease process in ADPKD is primarily influenced by the RAAS and endothelial cell dysfunction associated with polycystin. Alongside these, the sympathetic nervous system activation, NO, endothelin-1, and ADMA are likely to exert pivotal deleterious effects on the endothelium[150,151].

TMA encompasses a clinical and pathological syndrome where endothelial damage precipitates manifestations such as thrombocytopenia, microangiopathic hemolytic anemia, and renal injury. Complement-mediated endothelial damage has been implicated as the primary pathogenesis of TMA[152]. Complement inhibitor therapies such as eculizumab have been shown to reduce the levels of markers of ED such as thrombomodulin, VCAM-1, and soluble tumor necrosis factor receptor 1[153].

Lupus nephritis patients after kidney transplant have been shown to have pre-mature CV events, which stem from ED[154]. ED in lupus nephritis is not localized to the kidney, but is systemic. The pathophysiology of ED in this condition is due to autoantibodies, inflammatory mediators, immune complex deposition in the vasculature, complement activation, oxidative stress, hypertension, and usage of immunosuppressive drugs such as corticosteroids and CNIs[155]. Newer biological agents and reduced steroid usage can improve endothelial functions in lupus nephritis patients[156].

ED has also been demonstrated in IgA nephropathy[157], anti-neutrophilic cytoplasmic antibody related vasculitis[158], and reflux nephropathy[158].

Clinical vignette: Statin therapy improves both dyslipidemia and FMD in patients with nephrotic syndrome[159].

Bone mineral disease: CKD-mineral and bone disorder (CKD-MBD) emerges due to disturbances in calcium and phosphorus regulation, accompanied by elevated parathyroid hormone and fibroblast growth factor 23 (FGF23) levels, and reduced levels of 1,25-dihydroxyvitamin D. These disruptions lead to aberrant bone restructuring and calcification beyond the skeletal system, contributing significantly to mortality rates among affected patients[160]. Phosphate retention due to low estimated glomerular filtration rate exacerbates vascular calcification and oxidative stress, leading to ED. Elevated phosphate levels disrupt vascular calcification regulation, inducing arterial wall thickening and stiffness, while triggering the transformation of vascular smooth muscle cells into osteoblast-like cells[161]. Diabetic patients with CKD develop a typical MBD, namely, adynamic bone disease (low bone turnover state), known for accelerated vascular calcification[162]. However, the mechanisms of ED in a low turnover state are not clearly defined[161].

Kidney transplant recipients frequently encounter disruptions in phosphate homeostasis immediately following transplantation. This spectrum encompasses early-onset hypophosphatemia within the initial 3 months post-transplantation, progressing toward late-stage complications such as hyperphosphatemia, hyperparathyroidism, and increased FGF23 levels[163]. Vitamin D deficiency and elevated FGF23 levels are associated with ED[164]. Vitamin D deficiency is associated with elevated e-selectin levels[164]. Klotho, an anti-vascular aging and kidney protective protein, is implicated in MBD and CV diseases[165]. FGF23 decreases klotho levels, but klotho improves vitamin D levels. So, klotho by itself, and with vitamin D is protective for the endothelium. Vitamin D-FGF23-Klotho axis alterations are demonstrated in both kidney donors (after donation) and recipients[166]. RAAS inhibitors increase klotho levels[167].

Clinical vignette: Routine vitamin D supplementation to enhance endothelial function represents a promising area of research. The authors are part of a randomized controlled trial that looks at the change in FMD and endothelial biomarkers with vitamin D (cholecalciferol) supplementation in stable kidney transplant recipients.

Diet and nutrients: Diet is an important parameter that affects vascular aging[168]. A healthy diet is protective of the endothelium. The Mediterranean diet reduces oxidative stress and improves ED. A recent study showed that during a median follow-up of 5.4 years, higher adherence to the Mediterranean diet was associated with significantly lower risks of graft failure (HR = 0.68; 95%CI: 0.50-0.91), kidney function decline (HR = 0.68; 95%CI: 0.55-0.85), and graft loss (HR = 0.74; 95%CI: 0.63-0.88)[169]. The Mediterranean diet reduces the risk of developing NODAT[170]. In a randomized crossover trial of 22 stable kidney transplant recipients on RAAS blockade, a 6-week low-sodium diet (target: 50 mmol/24 h) significantly reduced systolic BP (from 140 to 129 mmHg) and diastolic BP (from 86 to 79 mmHg) compared to a regular-sodium diet (target: 150 mmol/24 h), without affecting urinary albumin excretion or estimated glomerular filtration rate[171]. Sodium restriction is thus effective for BP management in this population. The dietary approaches to stop hypertension (DASH) diet is characterized by a high intake of fruits, vegetables, legumes, nuts, and whole grains, along with low-fat dairy products. It emphasizes reduced consumption of sodium, red and processed meats, and sugar-sweetened beverages. In a prospective cohort study of 632 stable renal transplant recipients[172], a higher DASH score was associated with a reduced risk of renal function decline (HR = 0.57; 95%CI: 0.33-0.96; P = 0.03) and all-cause mortality (HR = 0.52; 95%CI: 0.32-0.83; P = 0.006) over a median follow-up of 5.3 years. Thus, both the Mediterranean and DASH diets may confer protective effects on endothelial function.

Isoflavone-containing soy products are protective of the endothelium. A small study of 20 patients with stable renal functions demonstrated improved FMD (3.2 ± 1.8% vs 6.3 ± 1.9%, respectively; P < 0.001) with 5 weeks of soy protein diet[173].

Clinical vignette: Intermittent fasting improves lipid profiles, decreases white adipose tissue, and enhances gut microbiome health. This leads to lower inflammation and a decreased risk of CV disease. Implementing intermittent fasting represents a compelling area of research in transplant settings for enhancing CV health.

Genomics: Personalized medicine for preventing CV events in kidney transplant recipients involves tailoring preventive and therapeutic strategies based on individual genetic, physiological, and clinical profiles. This approach integrates genetic risk assessments, such as the genetic risk score (GRS) derived from specific single nucleotide polymorphisms (SNPs), to identify patients at higher risk for CV complications. A composite GRS based on 27 SNPs, previously shown to predict CV events in the general population, demonstrated significant predictive value for major CV events in kidney transplant recipients (HR = 1.81; P = 0.006)[174]. This suggests that the GRS is applicable in renal transplant populations, with the potential for further refinement to enhance its predictive accuracy in this context. Polygenic risk scores from donors and recipients were able to predict the occurrence of NODAT[175]. The eNOS gene polymorphism identifies allograft outcomes related with atherosclerosis such as interstitial fibrosis and tubular atrophy[176].

Clinical vignette: Pharmacogenomics helps in precision prescribing and can avoid unnecessary CV side effects of immunosuppressants[177].

Figure 1 highlights the common pathways and risk factors leading to ED and major CV adverse events among post-kidney transplant recipients.

Figure 1 Common pathways and risk factors leading to endothelial dysfunction and major cardiovascular adverse events among post-kidney transplant recipients. AGEs: Advanced glycation end products; CKD: Chronic kidney disease; CVD: Cardiovascular diseases; FFA: Free fatty acids; FGF: Fibroblast growth factor; LVH: Left ventricular hypertrophy; NO: Nitric oxide; NODAT: New-onset diabetes after transplant; PTH: Parathyroid hormone; SES: Socioeconomic status.

METHODS TO STUDY ENDOTHELIAL FUNCTIONS

Biomarkers

Biomarkers provide diagnostic information and insights into newer diagnostics, pathogenesis, and therapeutic targets. Biomarkers of endothelial activation and dysfunction are valuable for diagnosing vascular pathologies. These biomarkers often lack specificity and sensitivity in assessing CV disease in humans. Clinical utility is hindered by factors like small sample sizes, conflicting data, and poor understanding of some of the functions of biomarkers. Despite these challenges, biomarkers offer insights into disease mechanisms and are promising research tools for CV diseases. A massive surge in the endothelial biomarkers research occurred with the emergence of COVID-19 infection, where identifying these biomarkers helped us to study endothelial biology in inflammatory conditions and better risk stratify patients based on CV health[178,179]. The European Society of Cardiology is advocating for the integration of biomarkers alongside FMD for better prediction of CV events[180,181]. Detailed reviews of biomarkers have previously been published[182-184]. Table 1 lists a few important biomarkers belonging to various functional groups.

Table 1 Important biomarkers of endothelial dysfunction belonging to various functional groups.

Functional group	Biomarkers
Oxidative stress	NADPH oxidase, ROS
Acute phase proteins	IL-1β, IL-6, TNF-α and its receptors, MCP-1, CRP, and SAA
Cell-adhesion molecules	ICAM-1, VCAM-1, e-selectin, p-selectin
Proteins modified by post-translational modification	OxLDL, OxHDL, carbamylated LDL, AGE, RAGE
Glycocalyx damage	Syndexan-1, endocan
Vascular tone	Endothelin-1, angiotensin II, resistin
Metabolizes catecholamines	Renalase
eNOS uncoupling	ADMA, SDMA
Thrombosis	PAI-1, angiopoietin-2, vWF, TF, soluble thrombomodulin, thromboxane A2, d-dimer, factor VIII, homocysteine
Marker of angiogenesis and hyperpermeability	VEGF-A, TGF-β1, PlGF
Marker of endothelial proliferation and apoptosis	Circulating endothelial cells
Membranous particles released from endothelial cells	Endothelial microvesicles
Endothelial-protective molecules	NO, klotho, sirtuins, Nrf2, PGI2
Others	miRNA, gut microbiota-derived metabolites, suPAR

ADMA: Asymmetric dimethylarginine; AGE: Advanced glycation end products; CRP: C-reactive protein; ICAM: Intercellular adhesion molecule; IL: Interleukin; MCP-1: Membrane cofactor protein-1; miRNA: Micro RNA; NADPH oxidase: Nicotinamide adenine dinucleotide oxidase; NO: Nitric oxide; Nrf2: Nuclear factor erythroid 2 related factor; OxLDL: Oxidized low-density lipoprotein; OxHDL: Oxidized high-density lipoprotein; PAI-1: Plasminogen activator inhibitor-1; PGI2: Prostaglandin I2; PlGF: Placental growth factor; RAGE: Receptors of advanced glycation end products; ROS: Reactive oxygen species; SAA: Serum amyloid A; SDMA: Symmetric dimethylarginine; suPAR: Soluble urokinase plasminogen activator receptor; TF: Tissue factor; TGF: Transforming growth factor; TNF: Tumor necrosis factor; VCAM: Vascular adhesion molecule; VEGF: Vascular endothelial growth factor; vWF: von Willebrand factor.

Vasomotor responses to determine endothelial functions

Arterial health is often assessed by examining the vessel’s capacity for dilation in response to various stimuli. Physiologically, a key marker of arterial health is the ability of arteries to undergo endothelium-dependent vasodilation in reaction to reactive hyperemia. This phenomenon occurs due to increased shear stress resulting from elevated blood flow. In healthy arteries, this dilatory response is mediated by the endothelium through the release of vasodilatory factors such as NO, which counteracts the elevated shear stress by promoting vessel relaxation. Thus, impaired or blunted vasodilation in response to reactive hyperemia can serve as an indicator of ED. Additionally, arterial dilation is also elicited through the action of endothelium-dependent vasodilators, such as acetylcholine, bradykinin, and serotonin[185]. These mediators trigger the release of NO, which induces arterial vasodilation by stimulating guanylate cyclase, which increases cyclic guanosine monophosphate levels, leading to smooth muscle relaxation. Exogenous NO donors such as glyceryl-trinitrate and smooth muscle vasodilators such as adenosine are used to measure non-endothelial-induced vasodilation[185]. Consequently, the methodologies employed are designed to evaluate the vasodilatory capacity of arteries in response to diverse physiological stimuli, providing critical insights into their functional integrity. Blood flow, in addition to modulating vascular tone, influences a range of physiological processes, including the regulation of endothelial barrier permeability, angiogenesis, endothelial cell proliferation, apoptosis, differentiation, senescence, metabolic activities, inflammatory responses, morphological changes, and extracellular matrix remodeling[186]. Thus, blood flow measurement helps in understanding endothelial functions[187].

Coronary endothelial function measurement

Invasive methods: Coronary ED is characterized by the lack of expected vasodilation and the inadequate increase in blood flow within the epicardial coronary arteries following acetylcholine administration. These changes are commonly assessed using two main techniques: Quantitative coronary angiography and intravascular ultrasound[185]. While definitions of an abnormal angiographic response may vary across sources, a practical criterion is any degree of constriction of the epicardial arteries exceeding 0%, but not exceeding 90% following acetylcholine administration[188]. Agents other than acetylcholine that can be used include bradykinin, papaverine, and substance P[189]. Changes in coronary blood flow (CBF), a surrogate of coronary microvascular functions, are assessed through coronary flow reserve (CFR), calculated as maximal CBF during induced hyperemia divided by resting CBF. A CFR below 2.0 indicates an abnormality. Invasive methods like doppler flow velocity or thermodilution are used, albeit with technical challenges and reproducibility concerns[185].

Non-invasive methods to study coronary microvascular functions include the following[190]: (1) Contrast echocardiography; (2) Transthoracic doppler echocardiography; (3) Contrast tomography (CT); (4) Positron emission tomography (PET); (5) Single-photon emission computed tomography; and (6) Cardiac magnetic resonance imaging. Among these imaging modalities, PET offers precise global and regional myocardial perfusion (MP), blood flow, and function measurements, with advanced software enabling routine assessment of myocardial blood flow and viability using tracers like 18F-fluoro deoxy-glucose and 82-rubidium[191]. Enhanced PET/CT scanners facilitate accurate coronary microvascular dysfunction evaluation and disease detection, with promising applications in identifying vulnerable plaques through atherosclerotic plaque imaging[191].

Assessing peripheral arterial endothelial functions

For asymptomatic patients, invasive techniques, as mentioned before, may not be suitable. Therefore, non-invasive methods to detect endothelial functions have become increasingly popular[192]. Assessment by some of the techniques has been shown to replicate the endothelial functions in the coronary circulation[193-195].

FMD: Skeletal muscle arteries respond rapidly to reactive hyperemia of the supplying muscles[196]. FMD of conduit arteries like the brachial artery is a commonly used test to measure endothelial function[197]. Brachial artery FMD, a marker of endothelium-dependent vasodilation in response to vascular hyperemia (created by deflating an already inflated cuff at supra systolic blood pressure), mirrors NO production in the endothelium and aligns with coronary artery endothelial function[198]. It has good reproducibility. A small study[199] evaluated the reliability and reproducibility of FMD and peripheral arterial tonometry (PAT) for assessing endothelial function. Simultaneous FMD (brachial artery ultrasound) and PAT (EndoPAT 2000) assessments were performed twice with a 30-minute interval and repeated after 2 days. Within-day variability was lower for FMD (10%) compared to PAT (18%; P < 0.05), while between-day variability was similar (11%). A significant correlation between PAT and FMD was observed (r = 0.57, P < 0.001). FMD showed better reliability for both within- and between-day measurements compared to PAT. Systematic reviews demonstrate that even a modest increase of 1% in FMD value is linked to a significant 12% to 13% reduction in the risk of CV events, regardless of traditional CV risk factors[200,201]. This underscores the potential of FMD as a valuable vascular biomarker for stratifying CV risk. FMD is measured by real-time computer-based software, which will detect changes in the vessel wall diameters in response to hyperemia compared to baseline, i.e., %FMD[202]. If not performed and analyzed consistently, FMD measurements can have high intra- and inter-observer variability. Standard reference ranges are available, which help identify and monitor at-risk individuals[198,203]. In 1579 healthy individuals, FMD was inversely correlated with age, body mass index, glucose, cholesterol, blood pressure, and brachial artery diameter. Receiver operating characteristic analysis indicated that FMD > 6.5% excludes coronary artery disease (95% sensitivity, 60% specificity), while FMD < 3.1% excludes 95% of healthy individuals[203]. A meta-analysis of 82 studies (n = 3509) found an average FMD of 6.4% (95%CI: 6.2%-6.7%) with a significant age-related decline (-0.3%/decade)[203].

Nitroglycerin-mediated dilatation: Nitroglycerine-induced vasodilation (NMD), a marker of endothelium-independent vasodilation, is used as a control test for FMD to confirm that impaired FMD is not due to vascular smooth muscle dysfunction or structural alterations[204]. Instead, it confirms that the observed FMD impairment genuinely stems from ED. Combined FMD and NMD can better predict CV events than NMD alone[205]. NMD is usually performed by administering sublingual isosorbide dinitrite[144]. Again, the reference values for NMD are available for the general population[198].

Venous occlusion plethysmography: Forearm blood flow changes are measured via plethysmography in response to vasoactive substances introduced through a cannulated brachial artery, allowing quantification of both endothelial-dependent and independent vasodilation. Because of its semi-invasiveness, time-consuming, and impracticality for serial measurements, it has limited usage in the current era[206].

PAT: PAT is an innovative approach for evaluating endothelial function, utilizing finger plethysmography to measure pulse wave amplitude (PWA) at rest and during shear stress[207]. The reactive hyperemia-PAT index quantifies the ratio of PWA signal post-cuff release to baseline via computerized algorithms[208]. PAT offers advantages like using the subject’s contralateral arm as an internal control and requiring minimal training. It correlates well with endothelial function across various populations and has predictive value for CV events[209,210].

Retinal artery diameters and reactivity: Evidence supports retinal microvascular analysis as a predictor of systemic CV risk and disease; however, this association is not yet strongly established[185]. Some studies highlight its clinical utility and predictive value for CV outcomes by assessing endothelial function, supporting its use in the primary and secondary prevention of chronic non-communicable diseases such as hypertension and diabetes mellitus[211,212]. Retinal vessel dimensions and flicker light-induced dilation are employed to detect endothelial functions[212]. Continuous retinal vessel diameter measurements during flicker light provocation reveal significant differences between those with CV disease and those with both CV disease + diabetes mellitus[212]. The distinct dilation and constriction patterns in diabetes mellitus (P < 0.030) could serve as a marker for monitoring CV disease progression[212]. The lack of randomized controlled studies and the non-availability of standard values hinder further progress in this field[185].

Studies on endothelial function testing in kidney transplant recipients: Kidney transplantation has been demonstrated to improve ED immediately after transplant, attributed to the reduction of uremia-related and traditional risk factors that cause endothelial damage[213-215]. The positive correlation measured in the above studies was seen until 90 days after the kidney transplant, as evidenced by improvement in FMD and biomarkers. However, their values were still significantly lower than in a healthy population[213]. In stable kidney transplant patients > 6 months after transplant, there was a significant decrease in FMD (-1.52% ± 2.74%; P = 0.03) at 3-6 months compared to baseline[216]. However, the decline in NMD was not significant. In a 6.5-year follow-up study of 152 kidney transplant recipients, significant post-transplant ED (defined as FMD ≤ 5.36%) was found to be associated with increased mortality (HR = 9.80; 95%CI: 1.29-74.62; P = 0.03) and higher risk of uncensored graft loss (HR = 7.80; 95%CI: 1.83-33.30; P = 0.01)[217]. Mortality risk increased with diminishing FMD levels until approximately 5% dilation, beyond which further reduction did not significantly affect the risk, suggesting a critical threshold for adverse outcomes in kidney transplant recipients[217]. Hence, a decline in FMD following an initial improvement is evident.

In a pilot study[218], MP reserve (MPR) was lower in kidney transplant patients (4.1 ± 1.1) compared to healthy controls (4.3 ± 1.6), but after adjusting for basal MP (1.3 ± 0.4 vs 1.0 ± 0.2 mL/minute/g) and cardiac workload, differences in MPR and stress MP (3.8 ± 1.0 vs 4.0 ± 0.9 mL/minute/g) were not significant. Increased resting MP in transplant patients is due to higher cardiac workload.

A study[219] analyzing endothelial biomarkers tried to delineate correlations between renalase and markers of inflammation and coagulation within a cohort of 62 kidney allograft recipients. Notably, renalase concentrations correlated with key endothelial markers such as vWF, thrombomodulin, ICAM-1, and VCAM-1[219]. Also, renalase was higher in hypertensive subjects compared to normotensive subjects[219]. ADMA (decreased eNOS) levels were associated with increased all-cause mortality (HR = 1.52; 95%CI: 1.26-1.83) in kidney transplant recipients[220].

CONCLUSION

ED in kidney transplant recipients is often under-recognized and inadequately treated, despite its significant CV implications. Increased CV mortality in this population is associated with ED. Many traditional and non-traditional risk factors for ED are modifiable, and optimal use of endothelial-protective medications is warranted. There is a critical need to include kidney transplant patients in CV clinical trials focusing on ED. Both FMD and biomarkers offer promise for early diagnosis of CV diseases compared to conventional diagnostic methods. As the number of kidney transplants continues to rise annually, emphasis should be placed on the prevention, early recognition, and treatment of ED to improve graft longevity and overall survival.