Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

CLIC-01: Manufacture and distribution of non-cryopreserved CAR-T cells for patients with CD19 positive hematologic malignancies.

Access to commercial CD19 CAR-T cells remains limited even in wealthy countries like Canada due to clinical, logistical, and financial barriers related to centrally manufactured products. We created a non-commercial academic platform for end-to-end manufacturing of CAR-T cells within Canada's publicly funded healthcare system. We report initial results from a single-arm, open-label study to determine the safety and efficacy of in-house manufactured CD19 CAR-T cells (entitled CLIC-1901) in participants with relapsed/refractory CD19 positive hematologic malignancies. Using a GMP compliant semi-automated, closed process on the Miltenyi Prodigy, T cells were transduced with lentiviral vector bearing a 4-1BB anti-CD19 CAR transgene and expanded. Participants underwent lymphodepletion with fludarabine and cyclophosphamide, followed by infusion of non-cryopreserved CAR-T cells. Thirty participants with non-Hodgkin's lymphoma (n=25) or acute lymphoblastic leukemia (n=5) were infused with CLIC-1901: 21 males (70%), median age 66 (range 18-75). Time from enrollment to CLIC-1901 infusion was a median of 20 days (range 15-48). The median CLIC-1901 dose infused was 2.3 × 106 CAR-T cells/kg (range 0.13-3.6 × 106/kg). Toxicity included ≥ grade 3 cytokine release syndrome (n=2) and neurotoxicity (n=1). Median follow-up was 6.5 months. Overall response rate at day 28 was 76.7%. Median progression-free and overall survival was 6 months (95%CI 3-not estimable) and 11 months (95% 6.6-not estimable), respectively. This is the first trial of in-house manufactured CAR-T cells in Canada and demonstrates that administering fresh CLIC-1901 product is fast, safe, and efficacious. Our experience may provide helpful guidance for other jurisdictions seeking to create feasible and sustainable CAR-T cell programs in research-oriented yet resource-constrained settings. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03765177, identifier NCT03765177.

A randomized control trial to assess the impact of vitamin D supplementation compared to placebo on vascular stiffness in chronic kidney disease patients.

BACKGROUND: Vitamin D deficiency is associated with cardiovascular (CV) risk in multiple populations, including those with chronic kidney disease (CKD). The active form of the hormone (1,25 OH2D3) binds to receptors in multiple organs. CKD patients are deficient in both 25 Vitamin D and 1,25 OH2D3. Clinical trial data demonstrating the benefits of vitamin D formulations are limited, and fail to show significant benefits on CV outcomes, and have compared different compounds, in various populations, and focused on a variety of outcomes. A understanding of the mechanism by which different vitamin D compounds confer CV protection in CKD is important for the design of future studies. METHODS/DESIGN: This 3 arm randomized prospective double-blinded placebo-controlled study examining the impact of calcitriol (1,25 OH2D3) and 25-hydroxyvitamin D3 supplementation compared to placebo on vascular stiffness, as measured by pulse wave velocity (PWV). Patients are enrolled from 2 tertiary care institutions if they meet inclusion criteria (stable estimated glomerular filtration rate (eGFR) between 15-45ml/min, <±5ml/min change in previous 6 months), on stable doses of renin-angiotensin aldosterone system blockade. For those already receiving vitamin D therapies, a 3 month washout period before randomization is mandatory. Treatment duration is 6 months; medications are given thrice weekly in fixed doses. The primary outcome measure is Vascular stiffness, measured non-invasively by pulse wave velocity (PWV). Other measurements include BP, kidney function and serial blood levels of biomarkers. The primary analysis will compare any vitamin D therapy versus placebo for the primary outcome defined as the change of PWV from baseline to 6 months. Analysis of covariance will be used to detect differences between vitamin D preparations in the magnitude of reduction in PWV. DISCUSSION: This study is novel in that we are using a robust study design in CKD patients (not on dialysis) comparing placebo to different forms of vitamin D supplementation in fixed doses, irrespective of baseline values. We hope to demonstrate the biological mechanistic effect of vitamin D supplementation on vascular function in order for this information to be used in designing larger randomized controlled trials. TRIAL REGISTRATION: Current Controlled Trials NCT01247311. Date of Registration: November 12, 2010.

Natural history of skeletal muscle mass changes in chronic kidney disease stage 4 and 5 patients: an observational study.

Cross-sectional studies in dialysis demonstrate muscle wasting associated with loss of function, increased morbidity and mortality. The relative drivers are poorly understood. There is a paucity of data regarding interval change in muscle in pre-dialysis and dialysis-dependant patients. This study aimed to examine muscle and fat mass change and elucidate associations with muscle wasting in advanced CKD. 134 patients were studied (60 HD, 28 PD, 46 CKD 4-5) and followed up for two years. Groups were similar in age, sex and diabetes prevalence. Soft tissue cross-sectional area (CSA) was measured annually on 3 occasions by a standardised multi-slice CT thigh. Potential determinants of muscle and fat CSA were assessed. Functional ability was assessed by sit-to-stand testing. 88 patients completed follow-up (40 HD, 16 PD, 32 CKD). There was a significant difference in percentage change in muscle CSA (MCSA) over year 1, dependant on treatment modality (χ(2) = 6.46; p = 0.039). Muscle loss was most pronounced in pre-dialysis patients. Muscle loss during year 1 was partially reversed in year 2 in 39%. Incident dialysis patients significantly lost MCSA during the year which they commenced dialysis, but not the subsequent year. Baseline MCSA, change in MCSA during year 1 and dialysis modality predicted year 2 change in MCSA (adjusted R(2) = 0.77, p<0.001). There was no correlation between muscle or fat CSA change and any other factors. MCSA correlated with functional testing, although MCSA change correlated poorly with change in functional ability. These data demonstrate marked variability in MCSA over 2 years. Loss of MCSA in both pre-dialysis and established dialysis patients is reversible. Factors previously cross-sectionally shown to correlate with MCSA did not correlate with wasting progression. The higher rate of muscle loss in undialysed CKD patients, and its reversal after dialysis commencement, suggests that conventional indicators may not result in optimal timing of dialysis initiation.

Responsiveness of FGF-23 and mineral metabolism to altered dietary phosphate intake in chronic kidney disease (CKD): results of a randomized trial.

BACKGROUND: High fibroblast growth factor-23 (FGF-23) levels are associated with adverse outcomes. We studied the responsiveness of FGF-23 and mineral metabolism to altered dietary phosphate intake in chronic kidney disease (CKD) and healthy control patients. METHODS: Thirty patients were enrolled: 18 normophosphatemic CKD subjects and 12 healthy controls. The study duration was 21 days with three 7-day dietary interventions; a high phosphate (HP, 2000 mg/day), low phosphate (750 mg/day) and low phosphate plus phosphate binder (aluminum hydroxide, 500 mg thrice daily with meals), with comparable macronutrient content, administered in random sequence. Baseline and weekly fasting morning measurements of FGF-23, serum phosphate (sPO(4)), 1,25-hydroxyvitamin D (1,25 D) and 24-h urinary calcium (uCa) and phosphate (uPO(4)) were collected. RESULTS: FGF-23 levels were higher in subjects versus controls (72 pg/mL versus 30 pg/mL) at baseline, while sPO(4) remained in the normal range throughout the study. The absolute changes of uPO(4) and uCa for CKD and controls vary according to diet. The absolute changes of FGF-23 and sPO(4) suggest that the effect of the diets might also depend on the CKD status (P-values interaction effect = 0.08 and 0.07, respectively); nonetheless, these changes are evident as a function of dietary interventions, irrespective of CKD status (P-values diet effect = 0.006 and <0.001, respectively). CONCLUSIONS: FGF-23 levels appear to be responsive to changes in diet in both CKD patients and controls. Further studies are required to determine whether lowering dietary phosphate and thus FGF-23 levels are of long-term benefit in CKD patients, irrespective of sPO(4) levels.

Vascular stiffness in incident peritoneal dialysis patients over time.

OBJECTIVE: Vascular stiffness is prevalent in end-stage renal disease patients and predicts adverse events. This study describes the prevalence of vascular stiffness and its associated factors in a cohort of incident peritoneal dialysis (PD) patients. METHODS: In a prospective observational study of 50 patients, carotid-femoral pulse wave velocity (PWV) were conducted at baseline, 3, 6 and 12 months after initiation of PD. Aortic calcification scores (ACS) were derived using plain lateral abdominal films. We examined the association of significant changes in PWV (defined as 1 m/s or 15% change from baseline) over 6 months in conjunction with demographic and clinical data. RESULTS: The mean age was 58 years, 67% were male, and 48% were Caucasian. One third was diabetic, and 23% had pre-existing cardiovascular disease. Median eGFR was 8.7 ml/ min. ACS was strongly correlated with PWV (r = 0.62, p < 0.0001). Over 6 months, 42% demonstrated significant increases, while 23% demonstrated decreases in their PWV. Factors shown to be associated with increasing PWV were Caucasian race (OR = 4.50; CI: 0.97 - 20.83), higher phosphate (OR = 8.36; CI: 1.10 - 63.51) and a lower baseline PWV (OR = 0.67; CI: 0.45 - 0.99). Decrease in PWV was associated with the absence of calcium based phosphate binder usage (OR = 0.11; CI: 0.02 - 0.73). Changes in weight and PWV at 12 months were significantly correlated (p = 0.007, r = 0.57). CONCLUSION: In this group of incident PD patients, we demonstrate a lower prevalence of vascular calcification than in hemodialysis patients, a correlation of calcification with PWV, and an important finding that PWV can change in either direction over a short period of time, which are associated with modifiable risk factors.

Angiopoietin-2 levels predict mortality in CKD patients.

BACKGROUND: The pathophysiology of aggravated atherosclerosis in chronic kidney disease (CKD) is still incompletely understood. However, there is an increasing focus on non-traditional risk factors, including endothelial dysfunction. Angiopoietin-2 (Ang-2) impairs endothelial function by inhibiting the binding of Angiopoietin-1 (Ang-1) to their shared receptor Tie2 and is increased in diabetes, hypertension, coronary heart disease and CKD. Furthermore, Ang-2 levels are associated with the prevalent vascular burden of CKD patients. Thus, we aimed to investigate its impact on outcome in CKD, the population most likely to die of cardiovascular events. METHODS: We prospectively studied 128 CKD patients [43 CKD Stage 4, 85 CKD Stage 5 (57 haemodialysis, 28 peritoneal dialysis)] over a follow-up period of 4 years. Biochemical and clinical parameters, including objective scoring of vascular calcification (VC) by computed tomography (CT) and arterial stiffness by applanation tonometry (including radial-dorsalis pedis pulse wave velocity (PWVrd)) were recorded. Baseline Ang-1 [enzyme-linked immunosorbent assay (ELISA)], Ang-2 [immunoluminometric assay (ILMA)] and soluble Tie2 (sTie2) (ELISA) levels were measured in this group as well as in 20 healthy controls. RESULTS: Ang-2 values were significantly higher in CKD patients than in controls (2.01 ± 0.94 versus 1.00 ± 0.47 ng/mL, P < 0.0001). Furthermore, Ang-2 was significantly higher in dialysis than in Stage 4 CKD patients and correlated with markers of vascular disease [cholesterol, hsCRP, osteoprotegerin (OPG)]. However, elevated Ang-2 was not associated with the degree of VC or with arterial stiffness. Cox-regression analysis detected Ang-2 as an independent predictor of mortality in both unadjusted [hazard ratio (HR) 1.15; P = 0.002] and models adjusted for age and VC (HR 1.14; P = 0.003). CONCLUSIONS: Ang-2 levels are associated with systemic markers/mediators of micro-inflammation in CKD patients. Furthermore, elevated Ang-2 levels are strong predictors of long-term mortality, independent of conduit arterial stiffness or VC.

Incident isolated 1,25(OH)(2)D(3) deficiency is more common than 25(OH)D deficiency in CKD.

INTRODUCTION: Vitamin D deficiencies are well described in general populations and in those with chronic kidney disease (CKD). Although serum 25(OH)D may be a good indicator of vitamin D status in healthy individuals, the hydroxylated product, 1,25(OH)(2)D, essential for important biological functions such as mineral metabolism, bone turnover, regulation of protein synthesis, cell differentiation and proliferation may be a more suitable indicator for individuals with CKD. METHODS: We report an observational prospective cohort study of the incidence after 12 months of new isolated 1,25(OH)(2)D and new 25(OH)D deficiency in CKD patients (estimated glomerular filtration rate [eGFR] <60 ml/min), who were vitamin D replete at baseline. All analyses were run in a central laboratory. RESULTS: Of 1,256 patients who completed the study at 12 months, 631 were replete in both 25(OH)D and 1,25(OH)(2)D at baseline; at 12 months, 65% remained replete, 25% developed an isolated 1,25(OH)(2)D deficiency, whereas only 6% developed an isolated 25(OH)D deficiency. Based on the multinomial logistic regression model, factors that were associated with 12-month changes in vitamin D status were diabetes, baseline values of eGFR, albumin and both 25(OH)D and 1,25(OH)(2)D (all p values <0.03). Patients with diabetes, lower albumin, lower eGFR, lower levels of 25(OH)D and 1,25(OH)(2)D at baseline were at increased risk of developing isolated 1,25(OH)(2)D deficiency. CONCLUSIONS: The high incidence of new isolated 1,25(OH)(2)D deficiency as compared with new 25(OH)D deficiency, in the presence of 25(OH)D sufficiency, brings into question the value of measuring 25(OH)D levels in CKD. The significance of these findings and implications for replacement strategies require further study.

Novel biomarkers do not correlate with severity of vascular stiffness in CKD patients with severe co-morbid disease.

BACKGROUND/AIMS: Novel biomarkers may help explain the pathobiology of vascular disease in chronic kidney disease, and thus set the stage for identification of therapeutic targets, potential reversibility, and improved outcomes in this population. METHODS: 124 subjects with GFR <60 ml/min or on renal replacement therapy underwent measurement of inflammatory, vascular and cardiac biomarkers as well as aortic pulse wave velocity (PWV) testing. A subset of patients (n = 60) had repeat PWV measured at 6 months. RESULTS: Thirty-four percent of the patients were diabetic, and 50% had a history of cardiovascular disease or congestive heart failure. Median PWV was 9.8 (IQR 8.3-12.7) m/s. No significant correlations between the measured biomarkers and baseline PWV was observed. An increase in PWV (>1.5 m/s) over 6 months was observed in those subjects with diabetes, a higher brain natriuretic peptide level, lower cholesterol and lower phosphate level. Age (HR 1.086, p = 0.0028), fetuin (0.024, p = 0.0448), and interleukin-10 (top tertile HR 4.720, p = 0.0359) were associated with mortality. CONCLUSIONS: In this cohort of patients with chronic kidney disease and diabetes and/or heart disease, we were unable to demonstrate that select biomarkers can inform processes leading to vascular disease. Biomarkers do appear to have utility in predicting future events in this population.

Circulating endotoxemia: a novel factor in systemic inflammation and cardiovascular disease in chronic kidney disease.

BACKGROUND AND OBJECTIVES: Translocated endotoxin derived from intestinal bacteria has a wide range of adverse effects on cardiovascular (CV) structure and function, driving systemic inflammation, atherosclerosis and oxidative stress. This study's aim was to investigate endotoxemia across the spectrum of chronic kidney disease (CKD). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Circulating endotoxin was measured in 249 patients comprising CKD stage 3 to 5 and a comparator cohort of hypertensive patients without significant renal impairment. Patients underwent extended CV assessment, including pulse wave velocity and vascular calcification. Hemodialysis (HD) patients also received detailed echocardiographic-based intradialytic assessments. Patients were followed up for 1 year to assess survival. RESULTS: Circulating endotoxemia was most notable in those with the highest CV disease burden (increasing with CKD stage), and a sharp increase was observed after initiation of HD. In HD patients, predialysis endotoxin correlated with dialysis-induced hemodynamic stress (ultrafiltration volume, relative hypotension), myocardial stunning, serum cardiac troponin T, and high-sensitivity C-reactive protein. Endotoxemia was associated with risk of mortality. CONCLUSIONS: CKD patients are characteristically exposed to significant endotoxemia. In particular, HD-induced systemic circulatory stress and recurrent regional ischemia may lead to increased endotoxin translocation from the gut. Resultant endotoxemia is associated with systemic inflammation, markers of malnutrition, cardiac injury, and reduced survival. This represents a crucial missing link in understanding the pathophysiology of the grossly elevated CV disease risk in CKD patients, highlighting the potential toxicity of conventional HD and providing a novel set of potential therapeutic strategies to reduce CV mortality in CKD patients.

Exploring the Relationships between Hemoglobin, the Endothelium and Vascular Health in Patients with Chronic Kidney Disease.

BACKGROUND/AIMS: The ideal hemoglobin target in chronic kidney disease remains unknown. Ultimately, individualized targets may depend upon the properties of the patient's endothelial and vascular milieu, and thus the complex relationships between these factors need to be further explored. METHODS: Forty-six patients with a glomerular filtration rate (GFR) <30 ml/min/1.73 m(2) or on renal replacement therapy underwent measurement of hemoglobin, endothelial microparticles (EMPs) and aortic pulse wave velocity (PWV) at 0, 3 and 6 months. In addition, a number of inflammatory, cardiac and vascular biomarkers were measured at baseline. RESULTS: No correlation was observed between baseline values of PWV and EMPs, PWV and hemoglobin, or hemoglobin and EMPs in the overall cohort. When stratified by CKD status, a positive correlation was observed between PWV and EMP CD41-/CD144+ in patients with GFR <30 ml/min/1.73 m(2) only (r = 0.54, p = 0.01). Asymmetric dimethylarginine correlated with baseline PWV (r = 0.27, p = 0.07), and remained significantly correlated with the 3- and 6-month PWV measurement. CONCLUSIONS: In this small heterogeneous cohort of dialysis and non-dialysis patients, we were unable to describe a physiologic link between anemia, endothelial dysfunction and arterial stiffness.

Arterial stiffness and functional properties in chronic kidney disease patients on different dialysis modalities: an exploratory study.

BACKGROUND: Abnormalities of vascular function and accumulation of oxidative stress have been associated with chronic kidney disease (CKD). Dialysis modalities, peritoneal dialysis (PD) and haemodialysis (HD) may differentially impact on vascular function and oxidative stress. METHODS: Patients undergoing living donor transplantation were studied for vascular stiffness using pulse wave velocity measurements, and inferior epigastric arteries were harvested to examine in vitro stiffness and functional properties and evidence of oxidative stress. Forty-one patients were studied representing PD (n = 12), HD (n = 14) and non-dialysed recipients (n = 15). RESULTS: We demonstrated differences in stiffness from in vivo and in vitro measurements such that non-dialysis < HD < PD groups. The stiffness measurements did not correlate with duration of CKD nor dialysis duration, but did so with phosphate levels (r = 0.356, P = 0.02). From the in vitro isometric force experiments, HD arteries demonstrated decreased contractility and endothelium-dependent relaxation compared with PD and non-dialysis vessels. Level of oxidative stress (as indicated by the 8-isoprostane level) was 30% higher in HD arteries than in PD arteries. Protein expression of inducible nitric oxide synthase, NADPH subunits and xanthine oxidase was upregulated in HD arteries, while superoxide dismutase was downregulated. The compromised vascular function in HD arteries was improved by pharmacological means that eliminated oxidative stress. CONCLUSIONS: We report associations between vasomotor function and oxidative stress in the vasculature of patients receiving different dialysis therapies. Oxidative stress, which may be differentially augmented during PD and HD, may play an important role in the vascular dysfunction in dialysis populations.

Differential microvasculature dysfunction in living kidney donor transplant recipients: nondialyzed versus dialyzed chronic kidney disease patients.

We hypothesized that there was differential vasomotor dysfunction in the microcirculation between nondialyzed and dialyzed chronic kidney disease (CKD) patients. During live donor kidney transplantation procedures, skin arterioles (SkA; internal diameter = 120 +/- 5 microm) from donors (n = 27) and recipients (nondialysis = 15; dialysis = 20) were dissected from the abdominal wall at the incision site. In vivo aortic pulse wave velocity (PWV) was also measured. In the in vitro isometric force measurement, nondialyzed SkA exhibited comparable contraction to donor SkA, whereas dialyzed SkA had 60 and 40-50% increase in contraction in response to depolarization and agonist (that is, phenylephrine, serotonin and endothelin-1) stimulation, respectively. The acetylcholine-induced relaxation in the nondialyzed SkA was decreased by 50% compared with dialyzed SkA. However, pre-incubation with superoxide dismutase greatly enhanced the relaxation response in the nondialyzed, but not in the dialyzed SkA and donor SkA. Pre-incubation with N(G)-nitro-L-arginine methyl ester (L-NAME) elevated the resting tension and left-shifted the concentration response curve of phenylephrine-stimulated contraction in the donor-SkA. L-NAME only increased the resting tension in the nondialyzed vessel. In vitro stiffness positively correlated with PWV (R(2) = 0.302, p = 0.001), and dialyzed SkA was 60% stiffer than nondialyzed and donor SkA. The acetylcholine relaxation was negatively correlated with PWV in donors and recipients (R(2) = 0.282, p = 0.01). In conclusion, we have uniquely demonstrated differential microvasculature dysfunction between nondialyzed and dialyzed CKD patients.

Systematic review of evidence for the use of intradialytic parenteral nutrition in malnourished hemodialysis patients.

OBJECTIVE: Intradialytic parenteral nutrition (IDPN) is widely used to treat malnourished hemodialysis (HD) patients. However, the benefits of this treatment are unknown. Moderate protein-energy malnutrition (PEM) is thought to affect 15% to 43% of maintenance HD patients, and is independently associated with mortality in this population. This study systematically reviews the current literature, to assess whether IDPN improves survival, quality of life, or nutritional status in those receiving maintenance HD. METHODS: Two investigators undertook a formal systematic review of the literature, using the following key search words: intradialytic parenteral nutrition or intradialytic total parenteral nutrition plus any combination of renal dialysis or kidney-failure or chronic kidney disease and parenteral nutrition or intravenous nutrition or intravenous feeding. RESULTS: The search identified three suitable randomized, controlled trials, only one of which investigated hard clinical endpoints. There were insufficient data to undertake a meta-analysis. CONCLUSIONS: The evidence from clinical studies is insufficient to demonstrate either a net benefit or a net harm associated with the providing IDPN to malnourished HD patients. We recommend that any patient in whom IDPN was deemed necessary be entered into a clinical trial or registry, to record hard clinical outcomes associated with the use of this treatment.

Plasma pyrophosphate and vascular calcification in chronic kidney disease.

BACKGROUND: Pyrophosphate (PPi) is a potent inhibitor of vascular calcification and may be deficient in renal failure. We sought to determine whether plasma PPi is affected by dialysis or the mode of dialysis and whether it correlates with vascular calcification. METHODS: PPi was measured in plasma samples stored from a recent study of vascular calcification in 54 HD patients, 23 peritoneal dialysis (PD) patients and 38 patients with stage 4 chronic kidney disease (CKD). Calcification was quantified in a standardized section of the superficial femoral artery using computed tomography, and PPi was measured by enzyme assay, at both baseline and 1 year. RESULTS: Baseline plasma PPi was weakly correlated with age and serum phosphate, but not with alkaline phosphatase activity or other biochemical parameters, and did not differ between HD, PD and CKD patients. Both baseline calcification score and change in the calcification score at 1 year decreased with increasing quartiles of plasma PPi. In a multivariate analysis, plasma PPi was independently correlated with baseline calcification (P = 0.039) and the change in calcification (P = 0.029). CONCLUSION: Plasma PPi is negatively associated with vascular calcification in end-stage renal disease (ESRD) and CKD but is not affected by dialysis, the mode of dialysis or nutritional or inflammatory status. Although these data are consistent with an inhibitory effect of PPi on vascular calcification, further studies are needed to establish a causal role.

Upregulation of matrix metalloproteinase-2 in the arterial vasculature contributes to stiffening and vasomotor dysfunction in patients with chronic kidney disease.

BACKGROUND: Cardiovascular disease is the leading cause of mortality in chronic kidney disease patients on maintenance dialysis. Given the importance of matrix metalloproteinase-2 (MMP-2) in matrix integrity, vascular cell function, and structural stability, we hypothesized that MMP-2 was elevated in the macrovasculature in dialyzed chronic kidney disease patients compared with chronic kidney disease patients not on dialysis and kidney donors. METHODS AND RESULTS: Arteries from live kidney donors (A(donor); n=30) and recipients (nondialysis [A(nondialyzed)], n=17; dialysis [A(dialyzed)], n=23 [peritoneal dialysis, n=10; hemodialysis, n=13]) were harvested during the transplantation procedure. Compared with A(donor), MMP-2 upregulation was evident in both recipient groups. Protein expression of latent plus active MMP-2 in A(dialyzed) was 2-fold that in A(nondialyzed). MMP-2 activity increased with length of dialysis (r=0.573, P=0.004). In A(dialyzed), medial elastic fiber fragmentation was pronounced, and the ratio of external elastic lamina to media was negatively correlated with MMP-2 activity (r=-0.638, P=0.001). A(dialyzed) was 25% stiffer than A(nondialyzed); this increased stiffness correlated with MMP-2 activity (r=0.728, P<0.0001) and the severity of medial calcium deposition (r=0.748, P=0.001). The contractile function and endothelium-dependent relaxation were reduced by 35% to 55% in A(dialyzed) and were negatively associated with MMP-2 activity (r=-0.608, P=0.002; r=-0.520, P=0.019, respectively). Preincubation with MMP-2 inhibitor significantly improved contractility and relaxation in A(dialyzed). CONCLUSIONS: We describe a strong correlation between MMP-2 activation and elastic fiber disorganization, stiffness, calcification, and vasomotor dysfunction in the arterial vasculature in dialyzed chronic kidney disease patients. These findings may contribute to an improved understanding of mechanisms important in vascular health in chronic kidney disease patients.

Matrix metalloproteinase-2 and -9 exacerbate arterial stiffening and angiogenesis in diabetes and chronic kidney disease.

AIMS: Chronic kidney disease (CKD) and diabetes are the prominent risk factors of cardiovascular disease (CVD). Matrix metalloproteinase (MMP)-2 and -9 regulate vascular structure by degrading elastic fibre and inhibit angiogenesis by generating angiostatin. We hypothesized that MMP-2 and -9 were up-regulated in the arterial vasculature from CKD patients with diabetes, compared with those without diabetes. METHODS AND RESULTS: During living donor transplantation procedures, arteries from donors (n = 8) and recipients (non-diabetic, n = 8; diabetic, n = 8; matched in age, gender, and dialysis treatments) were harvested. Diabetic arteries had increased MMP-2 and -9 activities by 42 and 116% compared with non-diabetic ones. Diabetic arteries were the stiffest, and the stiffness measurement was highly correlated with the summation of MMP-2 + MMP-9 activities (r = 0.738, P = 0.0002). Pulse wave velocity measurements correlated with MMP activity (r = 0.683, P = 0.005). Elastic fibre degradation and calcification were worst in diabetic vessels. The phosphate level, which was 25% higher in diabetic patients, correlated with MMP activity (r = 0.513, P = 0.04) and in vitro stiffness (r = 0.545, P = 0.03), respectively. Angiostatin expression was doubled, whereas vascular endothelial growth factor was 50% reduced in diabetic compared with non-diabetic vessels. Microvascular density in diabetic vessels was 48% of that in non-diabetic ones, and it was strongly associated with MMP activity (r = -0.792, P < 0.0001) and vasorelaxation (r = 0.685, P = 0.0009). CONCLUSION: Using a matched case-control design, we report up-regulation of MMP-2 and -9 in diabetic CKD arteries and correlate those with stiffening, impaired angiogenesis, and endothelial dysfunction. These findings may help to explain the high susceptibility of CVD in diabetic and non-diabetic CKD patients.

Elevated osteoprotegerin is associated with all-cause mortality in CKD stage 4 and 5 patients in addition to vascular calcification.

BACKGROUND: Cardiovascular disease is the leading cause of death in the chronic kidney disease (CKD) population. The mechanisms of vascular damage are not fully understood. The objective of this study was to prospectively investigate the importance of novel mediators of vascular damage, in conjunction with vascular calcification (VC), on survival. METHODS: A total of 134 subjects [60 haemodialysis (HD), 28 peritoneal dialysis (PD) and 46 CKD stage 4] were studied. All survivors completed 40 months of follow-up. VC was measured using multi-slice spiral CT of the superficial femoral artery. Circulating osteoprotegerin (OPG), Fetuin-A and high sensitivity C-reactive protein (hs-CRP) were measured in addition to standard clinical biochemical analysis. RESULTS: After a 40-month follow-up, 31 patients had died (27 men and 4 women). Of 31 subjects, 31 had evidence of significant VC. The majority of deaths were in the HD group (48%), 36% were PD subjects and 16% were CKD subjects. The outcome of interest was survival at the end of follow-up. Multivariate logistical regression analysis revealed male gender [OR 8.06 (1.34-48.450) P = 0.02], OPG >25 pmol/L [OR 5.31(1.35-20.88) P = 0.02] and hypoalbuminaemia [OR 0.26 (0.12-0.56) P < 0.01], were associated with increased odds of death. CONCLUSION: We have previously reported that VC and low albumin predict death in CKD stages 4 and 5 over a 2-year follow-up period. These data show that OPG, independent of CRP, is also associated with a negative outcome. The mechanisms remain to be elucidated; however, it is likely that they are associated with vascular damage through mechanisms in addition to VC.

Myocardial contractile function and intradialytic hypotension.

Dialysis-induced hypotension remains a significant problem in hemodialysis (HD) patients. Numerous factors result in dysregulation of blood pressure control and impaired myocardial reserve in response to HD-induced cardiovascular stress. Episodic intradialytic hypotension may be involved in the pathogenesis of evolving myocardial injury. We performed an initial pilot investigation of cardiovascular functional response to pharmacological cardiovascular stress in hypotension-resistant (HR) and hypotension-prone (HP) HD patients. We studied 10 matched chronic HD patients (5 HP, 5 HR). Dobutamine-atropine stress (DAS) was performed on a nondialysis short interval day, with noninvasive pulse-wave analysis using the Finometer to continuously measure hemodynamic variables. Baroreflex sensitivity was assessed at rest and during DAS. Baseline hemodynamic variables were not significantly different. The groups had differing hemodynamic responses to DAS. The Mean arterial pressure was unchanged in the HR group but decreased in HP patients (-13.6 +/- 3.5 mmHg; P<0.001). This was associated with failure to significantly increase cardiac output in the HP group (cf. increase in cardiac output in the HR group of +33.4 +/- 6%; P<0.05), and a reduced response in total peripheral resistance (HP -10.3 +/- 6.8%, HR -22.7 +/- 2.9%, P=NS). Baroreflex sensitivity was not significantly different between groups at baseline or within groups with increasing levels of DAS; however, the mean baroreflex sensitivity was higher in HR cf. HP subjects throughout pharmacological stress (P<0.05). Hypotension-prone patients appear to have an impaired cardiovascular response to DAS. The most significant abnormality is an impaired myocardial contractile reserve. Early identification of these patients would allow utilization of therapeutic strategies to improve intradialytic tolerability, potentially abrogating aggravation of myocardial injury.

Vascular calcification is associated with impaired microcirculatory function in chronic haemodialysis patients.

Although vascular calcification (VC) in dialysis patients is associated with increased cardiovascular events, the pathophysiology is still largely obscure. Microcirculatory dysfunction may contribute to demand myocardial ischaemia. We have studied cutaneous microcirculatory function in haemodialysis (HD) patients with and without large-vessel VC. 37 non-diabetic subjects (20 HD and 17 healthy controls) were studied. VC was assessed using CT scanning of a standardised segment of superficial femoral artery (11 VC+, 9 VC-). Laser Doppler imaging was undertaken using a Periscan PIM II(R) at rest and under vasodilator challenge. Baseline perfusion was not statistically different in VC+ patients than VC- patients or controls (1.03 +/- 0.2, 1.08 +/- 0.2, 0.93 +/- 0.3 PU respectively). Overall, the maximum vasodilatory response to both ACh (p < 0.001) and SNP (p = 0.004) was lower in the HD than the control group. In addition, the HD patients took longer to reach a maximum vasodilatation than the controls (p = 0.008 for ACh, n.s. for SNP). Further, the maximum vasodilatory response in the VC+ patients was lower and patients took longer to reach maximum vasodilatation than the VC- group. We have demonstrated, for the first time, impaired and dysregulated microcirculatory function in patients with VC. This may be important in understanding the pathophysiology of the complications and cardiovascular consequences of VC.