Genetic imputation of kidney transcriptome, proteome and multi-omics illuminates new blood pressure and hypertension targets.

Genetic mechanisms of blood pressure (BP) regulation remain poorly defined. Using kidney-specific epigenomic annotations and 3D genome information we generated and validated gene expression prediction models for the purpose of transcriptome-wide association studies in 700 human kidneys. We identified 889 kidney genes associated with BP of which 399 were prioritised as contributors to BP regulation. Imputation of kidney proteome and microRNAome uncovered 97 renal proteins and 11 miRNAs associated with BP. Integration with plasma proteomics and metabolomics illuminated circulating levels of myo-inositol, 4-guanidinobutanoate and angiotensinogen as downstream effectors of several kidney BP genes (SLC5A11, AGMAT, AGT, respectively). We showed that genetically determined reduction in renal expression may mimic the effects of rare loss-of-function variants on kidney mRNA/protein and lead to an increase in BP (e.g., ENPEP). We demonstrated a strong correlation (r = 0.81) in expression of protein-coding genes between cells harvested from urine and the kidney highlighting a diagnostic potential of urinary cell transcriptomics. We uncovered adenylyl cyclase activators as a repurposing opportunity for hypertension and illustrated examples of BP-elevating effects of anticancer drugs (e.g. tubulin polymerisation inhibitors). Collectively, our studies provide new biological insights into genetic regulation of BP with potential to drive clinical translation in hypertension.

Long-term results of autologous scaffold-free tissue-engineered vascular graft for hemodialysis access.

BACKGROUND: The growing size of the end stage renal disease (ESRD) population highlights the need for effective dialysis access. Exhausted native vascular access options have led to increased use of catheters and prosthetic shunts, which are both associated with high risks of access failure and infection. Emerging alternatives include tissue-engineered vascular grafts (TEVG). Here we present the endpoint results for 10 ESRD patients with the scaffold-free tissue-engineered vascular access produced from sheets of extracellular matrix produced in vitro by human cells in culture. METHODS: Grafts were implanted as arteriovenous shunts in 10 ESRD patients with a complex history of access failure. Follow-up included ultrasound control of graft morphology and function, dialysis efficiency, access failure, intervention rate, as well as immunohistochemical analysis of graft structure. RESULTS: One patient died of unrelated causes and three shunts failed to become useable access grafts during the 3-month maturation phase. The 12-month primary and secondary patency for the other six shunts was 86%. Survival of six shunts functioning as the vascular access was 22 ± 12 months with longest primary patency of 38.6 months. The dialysis event rate of 3.34 per patient-year decreased significantly with the use of this TEVG to 0.67. CONCLUSIONS: This living autologous tissue-engineered vascular graft seems to be an alternative to synthetic vascular access options, exhibiting advantages of native arteriovenous fistula.

First in Poland Simultaneous Liver-Lung Transplantation With Liver-First Approach for Recipient Due to Cystic Fibrosis: A Case Report.

Cystic fibrosis is an autosomal progressive disease affecting the lung, pancreas, and liver. Some patients develop end-stage respiratory and liver failure. For such patients, combined lung-liver transplantation remains the only therapeutic option. In this article we present the first simultaneous lung-liver transplantation in Poland, as well as in Central and Eastern Europe, with detailed clinical history, surgical aspects, and postoperative course.

Uncovering genetic mechanisms of hypertension through multi-omic analysis of the kidney.

The kidney is an organ of key relevance to blood pressure (BP) regulation, hypertension and antihypertensive treatment. However, genetically mediated renal mechanisms underlying susceptibility to hypertension remain poorly understood. We integrated genotype, gene expression, alternative splicing and DNA methylation profiles of up to 430 human kidneys to characterize the effects of BP index variants from genome-wide association studies (GWASs) on renal transcriptome and epigenome. We uncovered kidney targets for 479 (58.3%) BP-GWAS variants and paired 49 BP-GWAS kidney genes with 210 licensed drugs. Our colocalization and Mendelian randomization analyses identified 179 unique kidney genes with evidence of putatively causal effects on BP. Through Mendelian randomization, we also uncovered effects of BP on renal outcomes commonly affecting patients with hypertension. Collectively, our studies identified genetic variants, kidney genes, molecular mechanisms and biological pathways of key relevance to the genetic regulation of BP and inherited susceptibility to hypertension.

Hypertension and renin-angiotensin system blockers are not associated with expression of angiotensin-converting enzyme 2 (ACE2) in the kidney.

AIMS: Angiotensin-converting enzyme 2 (ACE2) is the cellular entry point for severe acute respiratory syndrome coronavirus (SARS-CoV-2)-the cause of coronavirus disease 2019 (COVID-19). However, the effect of renin-angiotensin system (RAS)-inhibition on ACE2 expression in human tissues of key relevance to blood pressure regulation and COVID-19 infection has not previously been reported. METHODS AND RESULTS: We examined how hypertension, its major metabolic co-phenotypes, and antihypertensive medications relate to ACE2 renal expression using information from up to 436 patients whose kidney transcriptomes were characterized by RNA-sequencing. We further validated some of the key observations in other human tissues and/or a controlled experimental model. Our data reveal increasing expression of ACE2 with age in both human lungs and the kidney. We show no association between renal expression of ACE2 and either hypertension or common types of RAS inhibiting drugs. We demonstrate that renal abundance of ACE2 is positively associated with a biochemical index of kidney function and show a strong enrichment for genes responsible for kidney health and disease in ACE2 co-expression analysis. CONCLUSION: Our results indicate that neither hypertension nor antihypertensive treatment is likely to alter the expression of the key entry receptor for SARS-CoV-2 in the human kidney. Our data further suggest that in the absence of SARS-CoV-2 infection, kidney ACE2 is most likely nephro-protective but the age-related increase in its expression within lungs and kidneys may be relevant to the risk of SARS-CoV-2 infection.

Synergistic effects of extracellular vesicle phenotyping and AFP in hepatobiliary cancer differentiation.

BACKGROUND: Biliary cancer, comprising cholangio- and gallbladder carcinomas, is associated with high mortality due to asymptomatic disease onset and resulting late diagnosis. Currently, no robust diagnostic biomarker is clinically available. Therefore, we explored the feasibility of extracellular vesicles (EVs) as a liquid biopsy tool for biliary cancer screening and hepatobiliary cancer differentiation. METHODS: Serum EVs of biliary cancer, hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer patients, as well as from healthy individuals, were isolated by sequential two-step centrifugation and presence of indicated EVs was evaluated by fluorescence activated cell sorting (FACS) analysis. RESULTS: Two directly tumour-related antigen combinations (AnnV+ CD44v6+ and AnnV+ CD44v6+ CD133+ ) and two combinations related to progenitor cells from the tumour microenvironment (AnnV+ CD133+ gp38+ and AnnV+ EpCAM+ CD133+ gp38+ ) were associated with good diagnostic performances that could potentially be used for clinical assessment of biliary cancer and differentiation from other cancer entities. With 91% sensitivity and 69% specificity AnnV+ CD44v6+ EVs showed the most promising results for differentiating biliary cancers from HCC. Moreover using a combined approach of EV levels of the four populations with serum AFP values, we obtained a perfect separation of biliary cancer and HCC with sensitivity, specificity, positive and negative predictive value all reaching 100% respectively. CONCLUSIONS: EV phenotyping, especially if combined with serum AFP, represents a minimally invasive, accurate liquid biopsy tool that could improve cancer screening and differential diagnosis of hepatobiliary malignancies.

Preoperative Neutrophil-to-Lymphocyte Ratio as an Independent Predictor of 1-Year Graft Loss and Mortality After Orthotopic Liver Transplantation.

INTRODUCTION: Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are low-cost and readily available inflammation markers. Previously, we revealed that the high preoperative neutrophil level is a recipient-related risk factor for the primary liver graft dysfunction (PGD), associated with a higher risk of early retransplantation or death. Here we aimed to evaluate the prognostic significance of preoperative neutrophil level, as well as the NLR and PLR in predicting a 1-year outcome of the orthotopic liver transplantation (OLTx). MATERIALS AND METHODS: One hundred and thirty-four patients who underwent the OLTx between 2012 and 2017 were enrolled. Analysis included, inter alia, etiology of liver failure and preoperative blood morphology. In the statistical analysis, the logistic regression model and receiver operator characteristic analysis were applied. RESULTS: In 1-year follow-up, 11% of patients died and 5% were retransplanted. Acute liver failure (ALF; odds ratio [OR] = 8.62, P = .007), autoimmune hepatitis (AIH; R = 5.25, P = .006), neutrophil level (OR = 1.23, P = .0003), MELD (OR = 1.05, P = .038), and the NLR (OR = 1.16, P = .001) were significant predictors of these detrimental outcomes. The multivariate analysis revealed etiology (AIH, P < .001 or ALF, P = .006) and NLR (P = .008) as the only independent predictors of 1-year graft loss or patient's death. Receiver operator characteristic analysis pointed at the NLR above 5.48 as their highly sensitive and specific risk factor. The PLR was not a prognostic biomarker. CONCLUSION: Achieved results call for further studies on the influence of the preoperative balance between systemic inflammation and immunity, expressed with the NLR on the long-term liver graft function.

Pentoxifylline and Methylprednisolone Additively Alleviate Kidney Failure and Prolong Survival of Rats after Renal Warm Ischemia-Reperfusion.

Renal ischemia-reperfusion injury (IRI) induces local inflammation leading to kidney damage. Since pentoxifylline (PTX) and steroids have distinct immunomodulatory properties, we aimed to evaluate for the first time their combined use in IRI-induced acute kidney injury (AKI) and chronic kidney disease (CKD) in rats. In two experiments, PTX (100 mg/kg body weight subcutaneously) was administered 90 min prior to renal IRI or/and methylprednisolone (MP; 100 mg/kg body weight intramuscularly) was infused 60 min after reperfusion of a solitary kidney (AKI model: 45 min ischemia, 48 male Sprague-Dawley rats) or one kidney with excision of contralateral kidney 2 weeks later (CKD model: 90 min ischemia, 38 rats). Saline was infused in place of PTX or/and MP depending on the group. Renal function (diuresis, serum creatinine, creatinine clearance, sodium and potassium excretion, and urine protein/creatinine) was assessed at 48 h and 120 h post-IRI (AKI model) or 4, 16 and 24 weeks after IRI, along with survival analysis (CKD model). More evidently at early stages of AKI or CKD, treated animals showed higher glomerular filtration and diminished tubular loss of electrolytes, more so with PTX + MP than PTX or MP (serum creatinine (µmol/L) at 48 h of AKI: 60.9 ± 19.1 vs. 131.1 ± 94.4 vs. 233.4 ± 137.0, respectively, vs. 451.5 ± 114.4 in controls, all p < 0.05; and at 4 weeks of CKD: 89.0 ± 31.9 vs. 118.1 ± 64.5 vs. 156.9 ± 72.6, respectively, vs. 222.9 ± 91.4 in controls, p < 0.05 for PTX or PTX + MP vs. controls and PTX + MP vs. MP). Survival was better by >2-fold with PTX + MP (89%) vs. controls (40%; p < 0.05). PTX + MP largely protect from IRI-induced AKI and CKD and subsequent mortality in rats. This calls for clinical investigations, especially in kidney transplantation.

Multipotency and cardiomyogenic potential of human adipose-derived stem cells from epicardium, pericardium, and omentum.

BACKGROUND: Acute myocardial infarction (MI) leads to an irreversible loss of proper cardiac function. Application of stem cell therapy is an attractive option for MI treatment. Adipose tissue has proven to serve as a rich source of stem cells (ADSCs). Taking into account the different morphogenesis, anatomy, and physiology of adipose tissue, we hypothesized that ADSCs from different adipose tissue depots may exert a diverse multipotency and cardiogenic potential. METHODS: The omental, pericardial, and epicardial adipose tissue samples were obtained from organ donors and patients undergoing heart transplantation at our institution. Human foreskin fibroblasts were used as the control group. Isolated ADSCs were analyzed for adipogenic and osteogenic differentiation capacity and proliferation potential. The immunophenotype and constitutive gene expression of alkaline phosphatase (ALP), GATA4, Nanog, and OCT4 were analyzed. DNA methylation inhibitor 5-azacytidine was exposed to the cells to stimulate the cardiogenesis. Finally, reprogramming towards cardiomyocytes was initiated with exogenous overexpression of seven transcription factors (ESRRG, GATA4, MEF2C, MESP1, MYOCD, TBX5, ZFPM2) previously applied successfully for fibroblast transdifferentiation toward cardiomyocytes. Expression of cardiac troponin T (cTNT) and alpha-actinin (Actn2) was analyzed 3 weeks after initiation of the cardiac differentiation. RESULTS: The multipotent properties of isolated plastic adherent cells were confirmed with expression of CD29, CD44, CD90, and CD105, as well as successful differentiation toward adipocytes and osteocytes; with the highest osteogenic and adipogenic potential for the epicardial and omental ADSCs, respectively. Epicardial ADSCs demonstrated a lower doubling time as compared with the pericardium and omentum-derived cells. Furthermore, epicardial ADSCs revealed higher constitutive expression of ALP and GATA4. Increased Actn2 and cTNT expression was observed after the transduction of seven reprogramming factors, with the highest expression in the epicardial ADSCs, as compared with the other ADSC subtypes and fibroblasts. CONCLUSIONS: Human epicardial ADSCs revealed a higher cardiomyogenic potential as compared with the pericardial and omental ADSC subtypes as well as the fibroblast counterparts. Epicardial ADSCs may thus serve as the valuable subject for further studies on more effective methods of adult stem cell differentiation toward cardiomyocytes.

First human use of an allogeneic tissue-engineered vascular graft for hemodialysis access.

An arteriovenous fistula is the current gold standard for chronic hemodialysis access. Tunneled catheters or synthetic grafts have poorer outcomes and much higher risks of infection. This report presents the first clinical use of a completely biological, allogeneic, nonliving, and human tissue-engineered vascular graft. Tissue-engineered vascular grafts built from allogeneic fibroblasts were implanted as shunts in three hemodialysis patients. The tissue-engineered vascular graft was stored for 9 months, without loss of mechanical strength. Implanted grafts showed no signs of degradation or dilation, with time points up to 11 months. Results of panel-reactive antibody and cross-reactivity tests showed no evidence of immune responses.

New biological solutions for hemodialysis access.

Since Scribner described the first prosthetic chronic dialysis shunt in 1961, the surgical techniques and strategies to maintain vascular access have improved dramatically. Today, hundreds of thousands of patients worldwide are treated with some combination of native vein fistula, synthetic vascular graft, or synthetic semipermanent catheter. Despite significantly lower efficacy compared with autologous fistulae, the basic materials used for synthetic shunts and catheters have evolved surprisingly slowly. The disparity between efficacy rates and concomitant maintenance costs has driven a strong campaign to decrease the use of synthetic grafts and catheters in favor of native fistulae. Whether arguing the benefits of Fistula First or "Catheter Last," the fact that clinicians are in need of an alternative to expanded polytetrafluoroethylene (ePTFE) is irrefutable. The poor performance of synthetic materials has a significant economic impact as well. End-stage renal disease (ESRD) accounts for approximately 6% of Medicare's overall budget, despite a prevalence of about 0.17%. Of that, 15%-25% is spent on access maintenance, making hemodialysis access a critical priority for Medicare. This clinical and economic situation has spawned an aggressive effort to improve clinical care strategies to reduce overall cost and complications. While the bulk of this effort has historically focused on developing new synthetic biomaterials, more recently, investigators have developed a variety of cell-based strategies to create tissue-engineered vascular grafts. In this article, we review the evolution of the field of cardiovascular tissue engineering. We also present an update on the Lifeline™ vascular graft, an autologous, biological, and tissue-engineered vascular graft, which was the first tissue-engineered graft to be used clinically in dialysis patients.

Case study: first implantation of a frozen, devitalized tissue-engineered vascular graft for urgent hemodialysis access.

Previously we reported on the mid- to long-term follow-up in the first clinical trial to use a completely autologous tissue-engineered graft in the high pressure circulation. In these early studies, living grafts were built from autologous fibroblasts and endothelial cells obtained from small skin and vein biopsies. The graft was assembled using a technique called tissue-engineering by self-assembly (TESA), where robust conduits were grown without support from exogenous biomaterials or synthetic scaffolding. One limitation with this earlier work was the long lead times required to build the completely autologous vascular graft. Here we report the first implant of a frozen, devitalized, completely autologous Lifeline™ vascular graft. In a departure from previous studies, the entire fibroblast layer, which provides the mechanical backbone of the graft, was air-dried then stored at -80°C until shortly before implant. Five days prior to implant, the devitalized conduit was rehydrated, and its lumen was seeded with living autologous endothelial cells to provide an antithrombogenic lining. The graft was implanted as an arteriovenous shunt between the brachial artery and the axillary vein in a patient who was dependent upon a semipermanent dialysis catheter placed in the femoral vein. Eight weeks postoperatively, the graft functions without complication. This strategy of preemptive skin and vein biopsy and cold-preserving autologous tissue allows the immediate availability of an autologous arteriovenous fistula, and is an important step forward in our strategy to provide allogeneic tissue-engineered grafts available "off-the-shelf".

Effectiveness of haemodialysis access with an autologous tissue-engineered vascular graft: a multicentre cohort study.

BACKGROUND: Application of a tissue-engineered vascular graft for small-diameter vascular reconstruction has been a long awaited and much anticipated advance for vascular surgery. We report results after a minimum of 6 months of follow-up for the first ten patients implanted with a completely biological and autologous tissue-engineered vascular graft. METHODS: Ten patients with end-stage renal disease who had been receiving haemodialysis through an access graft that had a high probability of failure, and had had at least one previous access failure, were enrolled from centres in Argentina and Poland between September, 2004, and April, 2007. Completely autologous tissue-engineered vascular grafts were grown in culture supplemented with bovine serum, implanted as arteriovenous shunts, and assessed for both mechanical stability during the safety phase (0-3 months) and effectiveness after haemodialysis was started. FINDINGS: Three grafts failed within the safety phase, which is consistent with failure rates expected for this high-risk patient population. One patient was withdrawn from the study because of severe gastrointestinal bleeding shortly before implantation, and another died of unrelated causes during the safety period with a patent graft. The remaining five patients had grafts functioning for haemodialysis 6-20 months after implantation, and a total of 68 patient-months of patency. In these five patients, only one intervention (surgical correction) was needed to maintain secondary patency. Overall, primary patency was maintained in seven (78%) of the remaining nine patients 1 month after implantation and five (60%) of the remaining eight patients 6 months after implantation. INTERPRETATION: Our proportion of primary patency in this high-risk cohort approaches Dialysis Outcomes Quality Initiative objectives (76% of patients 3 months after implantation) for arteriovenous fistulas, averaged across all patient populations.

Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery.

We have previously reported the initial clinical feasibility with our small diameter tissue engineered blood vessel (TEBV). Here we present in vitro results of the mechanical properties of the TEBVs of the first 25 patients enrolled in an arterio-venous (A-V) shunt safety trial, and compare these properties with those of risk-matched human vein and artery. TEBV average burst pressures (3490+/-892 mmHg, n=230) were higher than native saphenous vein (SV) (1599+/-877 mmHg, n=7), and not significantly different from native internal mammary artery (IMA) (3196+/-1264 mmHg, n=16). Suture retention strength for the TEBVs (152+/-50 gmf) was also not significantly different than IMA (138+/-50 gmf). Compliance for the TEBVs prior to implantation (3.4+/-1.6%/100 mmHg) was lower than IMA (11.5+/-3.9%/100 mmHg). By 6 months post-implant, the TEBV compliance (8.8+/-4.2%/100 mmHg, n=5) had increased to values comparable to IMA, and showed no evidence of dilation or aneurysm formation. With clinical time points beyond 21 months as an A-V shunt without intervention, the mechanical tests and subsequent lot release criteria reported here would seem appropriate minimum standards for clinical use of tissue engineered vessels.

Selected climatic variables and blood pressure in Central European patients with chronic renal failure on haemodialysis treatment.

UNLABELLED: BACKGROUND/AIMS;Higher blood pressure (BP) in winter has been documented in healthy and hypertensive adults. It may potentially contribute to the observed excess winter cardiovascular mortality in the general population. The aim of the study was to assess whether BP varies similarly among patients with chronic renal failure on haemodialysis treatment, who present an increased risk of cardiovascular death. METHODS: We retrospectively analysed values of pre-dialysis BP and parameters of fluid retention--pre-dialysis body weight and inter-dialytic weight gain measured in 49 patients (23 male, 26 female; aged 46.0+/-13.5 years) from 1995 to 1998. For each patient we calculated deviations of monthly mean values of systolic BP, diastolic BP, pre-dialysis body weight and inter-dialytic weight gain from the lowest monthly means of these parameters in a given year. Monthly means of these deviations for the whole study group (dSBP, dDBP, dBW, dWG, respectively) were subsequently computed. Monthly means of air temperature (T), air relative humidity (H) and atmospheric pressure (AP) were provided by the local Institute of Meteorology. The Wilcoxon paired test was applied to compare mean values of BPs and parameters of fluid retention of every patient in three warmest and three coldest months of each year. Spearman rank correlation analysis was employed to evaluate relationships between dSBP, dDBP and climatic variables, dBW or dWG. RESULTS: Systolic BP was higher in summer than in winter (146.6+/-20.5 vs 143.4+/-18.9 mmHg; p<0.00001). Diastolic BP was also higher in summer than in winter (82.6+/-8.5 vs 79.6+/-7.3 mmHg; p<10(-9)). Pre-dialysis body weight and inter-dialytic weight gain did not differ between summer and winter (66.0+/-13.2 vs 66.0+/-13.2 kg; p=0.98 and 2.27+/-0.6 vs 2.29+/-0.5 kg; p=0.53). There was a positive correlation between dSBP and T (RS=0.424, p<0.003), as well as dDBP and T (RS=0.591, p<0.00001) and an inverse correlation between dSBP and H (RS=-0.372, p<0.01), as well as dDBP and H (RS=-0.408, p<0.004). There were no significant associations between BPs and AP, dBW or dWG. CONCLUSIONS: In haemodialysed patients from southern Poland, BP is higher in summer than in winter. Changes in BP are related to seasonal changes in climatic variables--air temperature and air relative humidity. Seasonal variation in BP is not associated with variation in fluid retention. Possible alteration of cardiovascular reactivity to changes in climatic environment in haemodialysed chronic renal failure patients may be one of the potential explanations of these observations.