Machine-perfused donor kidneys as a source of human renal endothelial cells.

Renal endothelial cells (ECs) play crucial roles in vasorelaxation, ultrafiltration, and selective transport of electrolytes and water, but also in leakage of the glomerular filtration barrier and inflammatory processes like complement activation and leukocyte recruitment. In addition, they are target cells for both cellular and antibody-mediated rejection in the transplanted kidney. To study the molecular and cellular processes underlying EC behavior in renal disease, well-characterized primary renal ECs are indispensible. In this report, we describe a straightforward procedure to isolate ECs from the perfusion fluid of human donor kidneys by a combination of negative selection of monocytes/macrophages, positive selection by CD31 Dynabeads, and propagation in endothelium-specific culture medium. Thus, we isolated and propagated renal ECs from 102 donor kidneys, representative of all blood groups and major human leukocyte antigen (HLA) class I and II antigens. The obtained ECs were positive for CD31 and von Willebrand factor, expressed other endothelial markers such as CD34, VEGF receptor-2, TIE2, and plasmalemmal vesicle associated protein-1 to a variable extent, and were negative for the monocyte marker CD14 and lymphatic endothelial marker podoplanin. HLA class II was either constitutively expressed or could be induced by interferon-γ. Furthermore, as a proof of principle, we showed the diagnostic value of this renal endothelial biobank in renal endothelium-specific cross-matching tests for HLA antibodies.NEW & NOTEWORTHY We describe a new and widely accessible approach to obtain human primary renal endothelial cells in a standardized fashion, by isolating from the perfusate of machine-perfused donor kidneys. Characterization of the cells showed a mixed population originating from different compartments of the kidney. As a proof of principle, we demonstrated a possible diagnostic application in an endothelium-specific cross-match. Next to transplantation, we foresee further applications in the field renal endothelial research.

Association of plasma somatostatin with disease severity and progression in patients with autosomal dominant polycystic kidney disease.

BACKGROUND: Somatostatin (SST) inhibits intracellular cyclic adenosine monophosphate (cAMP) production and thus may modify cyst formation in autosomal dominant polycystic kidney disease (ADPKD). We investigated whether endogenous plasma SST concentration is associated with disease severity and progression in patients with ADPKD, and whether plasma SST concentrations change during treatment with a vasopressin V2 receptor antagonist or SST analogue. METHODS: In this observational study, fasting concentrations of SST were measured in 127 ADPKD patients (diagnosed upon the revised Ravine criteria) by ELISA. cAMP was measured in 24 h urine by Radio Immuno Assay. Kidney function was measured (mGFR) as 125I-iothalamate clearance, and total kidney volume was measured by MRI volumetry and adjusted for height (htTKV). Disease progression was expressed as annual change in mGFR and htTKV. Additionally, baseline versus follow-up SST concentrations were compared in ADPKD patients during vasopressin V2 receptor antagonist (tolvaptan) (n = 27) or SST analogue (lanreotide) treatment (n = 25). RESULTS: In 127 ADPKD patients, 41 ± 11 years, 44% female, eGFR 73 ± 32 ml/min/1.73m2, mGFR 75 ± 32 ml/min/1.73m2 and htTKV 826 (521-1297) ml/m, SST concentration was 48.5 (34.3-77.8) pg/ml. At baseline, SST was associated with urinary cAMP, mGFR and htTKV (p = 0.02, p = 0.004 and p = 0.02, respectively), but these associations lost significance after adjustment for age and sex or protein intake (p = 0.09, p = 0.06 and p = 0.15 respectively). Baseline SST was not associated with annual change in mGFR, or htTKV during follow-up (st. ß = - 0.02, p = 0.87 and st. ß = - 0.07, p = 0.54 respectively). During treatment with tolvaptan SST levels remained stable 38.2 (23.8-70.7) pg/mL vs. 39.8 (31.2-58.5) pg/mL, p = 0.85), whereas SST levels decreased significantly during treatment with lanreotide (42.5 (33.2-55.0) pg/ml vs. 29.3 (24.8-37.6), p = 0.008). CONCLUSIONS: Fasting plasma SST concentration is not associated with disease severity or progression in patients with ADPKD. Treatment with lanreotide caused a decrease in SST concentration. These data suggest that plasma SST cannot be used as a biomarker to assess prognosis in ADPKD, but leave the possibility open that change in SST concentration during lanreotide treatment may reflect therapy efficacy.

Crotamine/siRNA Nanocomplexes for Functional Downregulation of Syndecan-1 in Renal Proximal Tubular Epithelial Cells.

Proteinuria drives progressive tubulointerstitial fibrosis in native and transplanted kidneys, mainly through the activation of proximal tubular epithelial cells (PTECs). During proteinuria, PTEC syndecan-1 functions as a docking platform for properdin-mediated alternative complement activation. Non-viral gene delivery vectors to target PTEC syndecan-1 could be useful to slow down alternative complement activation. In this work, we characterize a PTEC-specific non-viral delivery vector composed of the cell-penetrating peptide crotamine complexed with a syndecan-1 targeting siRNA. Cell biological characterization was performed in the human PTEC HK2 cell line, using confocal microscopy, qRT-PCR, and flow cytometry. PTEC targeting in vivo was carried out in healthy mice. Crotamine/siRNA nanocomplexes are positively charged, about 100 nm in size, resistant to nuclease degradation, and showed in vitro and in vivo specificity and internalization into PTECs. The efficient suppression of syndecan-1 expression in PTECs mediated by these nanocomplexes significantly reduced properdin binding (p < 0.001), as well as the subsequent complement activation by the alternative complement pathway (p < 0.001), as observed in either normal or activated tubular conditions. To conclude, crotamine/siRNA-mediated downregulation of PTEC syndecan-1 reduced the activation of the alternative complement pathway. Therefore, we suggest that the present strategy opens new venues for targeted proximal tubular gene therapy in renal diseases.

Iron deficiency is related to lower muscle mass in community-dwelling individuals and impairs myoblast proliferation.

BACKGROUND: Loss of muscle mass is linked with impaired quality of life and an increased risk of morbidity and premature mortality. Iron is essential for cellular processes such as energy metabolism, nucleotide synthesis and numerous enzymatic reactions. As the effects of iron deficiency (ID) on muscle mass and function are largely unknown, we aimed to assess the relation between ID and muscle mass in a large population-based cohort, and subsequently studied effects of ID on cultured skeletal myoblasts and differentiated myocytes. METHODS: In a population-based cohort of 8592 adults, iron status was assessed by plasma ferritin and transferrin saturation, and muscle mass was estimated using 24-h urinary creatinine excretion rate (CER). The relationships of ferritin and transferrin saturation with CER were assessed by multivariable logistic regression. Furthermore, mouse C2C12 skeletal myoblasts and differentiated myocytes were subjected to deferoxamine with or without ferric citrate. Myoblast proliferation was measured with a colorimetric 5-bromo-2'-deoxy-uridine ELISA assay. Myocyte differentiation was assessed using Myh7-stainings. Myocyte energy metabolism, oxygen consumption rate and extracellular acidification rate were assessed using Seahorse mitochondrial flux analysis, and apoptosis rate with fluorescence-activated cell sorting. RNA sequencing (RNAseq) was used to identify ID-related gene and pathway enrichment in myoblasts and myocytes. RESULTS: Participants in the lowest age- and sex-specific quintile of plasma ferritin (OR vs middle quintile 1.62, 95% CI 1.25-2.10, P < 0.001) or transferrin saturation (OR 1.34, 95% CI 1.03-1.75, P = 0.03) had a significantly higher risk of being in the lowest age- and sex-specific quintile of CER, independent of body mass index, estimated GFR, haemoglobin, hs-CRP, urinary urea excretion, alcohol consumption and smoking status. In C2C12 myoblasts, deferoxamine-induced ID reduced myoblast proliferation rate (P-trend <0.001) but did not affect differentiation. In myocytes, deferoxamine reduced myoglobin protein expression (-52%, P < 0.001) and tended to reduce mitochondrial oxygen consumption capacity (-28%, P = 0.10). Deferoxamine induced gene expression of cellular atrophy markers Trim63 (+20%, P = 0.002) and Fbxo32 (+27%, P = 0.048), which was reversed by ferric citrate (-31%, P = 0.04 and -26%, P = 0.004, respectively). RNAseq indicated that both in myoblasts and myocytes, ID predominantly affected genes involved in glycolytic energy metabolism, cell cycle regulation and apoptosis; co-treatment with ferric citrate reversed these effects. CONCLUSIONS: In population-dwelling individuals, ID is related to lower muscle mass, independent of haemoglobin levels and potential confounders. ID impaired myoblast proliferation and aerobic glycolytic capacity, and induced markers of myocyte atrophy and apoptosis. These findings suggest that ID contributes to loss of muscle mass.

Circulating FGF21 Concentration, Fasting Plasma Glucose, and the Risk of Type 2 Diabetes: Results From the PREVEND Study.

OBJECTIVE: Fibroblast growth factor 21 (FGF21) is a peptide hormone synthesized by several organs and regulates, among others, energy homeostasis. In obesity, insulin resistance and type 2 diabetes (T2D), higher circulating FGF21 concentrations have been found. Temporal analyses in murine studies demonstrate that FGF21 increases before insulin resistance occurs. The current study aims to investigate in time-to-event analyses whether FGF21 may be an early biomarker in the development of T2D. RESEARCH DESIGN AND METHODS: Circulating FGF21 was measured using an immunoassay of the Mesoscale U-PLEX assay platform. The study outcome was incident T2D. Associations of circulating FGF21 concentration with T2D were quantified using Cox proportional hazards models with adjustments for potential confounders. RESULTS: We included 5244 participants aged 52 ± 12 years, of whom 50% were male. Median [interquartile range] circulating FGF21 concentration was 860 [525-1329] pg/mL. During 7.3 [6.1-7.7] years of follow-up, 299 (5.7%) participants developed T2D. In fully adjusted analyses, higher circulating FGF21 concentration was associated with an increased risk of incident T2D (hazard ratio per doubling: 1.26 [95% CI, 1.06-1.51]; P = 0.008), with effect modification by fasting plasma glucose, consistent with strengthening of the association at lower fasting glucose (interaction coefficient: -0.12; P = 0.022). CONCLUSION: Higher circulating FGF21 concentrations are independently associated with an increased risk of incident T2D in participants with a low fasting plasma glucose, making circulating FGF21 concentration a potential early biomarker for type 2 diabetes.

Fasting Proinsulin Independently Predicts Incident Type 2 Diabetes in the General Population.

Fasting proinsulin levels may serve as a marker of ß-cell dysfunction and predict type 2 diabetes (T2D) development. Kidneys have been found to be a major site for the degradation of proinsulin. We aimed to evaluate the predictive value of proinsulin for the risk of incident T2D added to a base model of clinical predictors and examined potential effect modification by variables related to kidney function. Proinsulin was measured in plasma with U-PLEX platform using ELISA immunoassay. We included 5001 participants without T2D at baseline and during a median follow up of 7.2 years; 271 participants developed T2D. Higher levels of proinsulin were associated with increased risk of T2D independent of glucose, insulin, C-peptide, and other clinical factors (hazard ratio (HR): 1.28; per 1 SD increase 95% confidence interval (CI): 1.08-1.52). Harrell's C-index for the Framingham offspring risk score was improved with the addition of proinsulin (p = 0.019). Furthermore, we found effect modification by hypertension (p = 0.019), eGFR (p = 0.020) and urinary albumin excretion (p = 0.034), consistent with an association only present in participants with hypertension or kidney dysfunction. Higher fasting proinsulin level is an independent predictor of incident T2D in the general population, particularly in participants with hypertension or kidney dysfunction.

Direct Evidence of Endothelial Dysfunction and Glycocalyx Loss in Dermal Biopsies of Patients With Chronic Kidney Disease and Their Association With Markers of Volume Overload.

Cardiovascular morbidity is a major problem in patients with chronic kidney disease (CKD) and endothelial dysfunction (ED) is involved in its development. The luminal side of the vascular endothelium is covered by a protective endothelial glycocalyx (eGC) and indirect evidence indicates eGC loss in CKD patients. We aimed to investigate potential eGC loss and ED in skin biopsies of CKD patients and their association with inflammation and volume overload. During living kidney transplantation procedure, abdominal skin biopsies were taken from 11 patients with chronic kidney disease stage 5 of whom 4 were treated with hemodialysis and 7 did not receive dialysis treatment. Nine healthy kidney donors served as controls. Biopsies were stained and quantified for the eGC marker Ulex europaeus agglutinin-1 (UEA1) and the endothelial markers vascular endothelial growth factor-2 (VEGFR2) and von Willebrand factor (vWF) after double staining and normalization for the pan-endothelial marker cluster of differentiation 31. We also studied associations between quantified log-transformed dermal endothelial markers and plasma markers of inflammation and hydration status. Compared to healthy subjects, there was severe loss of the eGC marker UEA1 (P < 0.01) while VEGFR2 was increased in CKD patients, especially in those on dialysis (P = 0.01). For vWF, results were comparable between CKD patients and controls. Skin water content was identical in the three groups, which excluded dermal edema as an underlying cause in patients with CKD. The dermal eGC/ED markers UEA1, VEGFR2, and vWF all associated with plasma levels of NT-proBNP and sodium (all R 2 > 0.29 and P < 0.01), except for vWF that only associated with plasma NT-proBNP. This study is the first to show direct histopathological evidence of dermal glycocalyx loss and ED in patients with CKD. In line with previous research, our results show that ED associates with markers of volume overload arguing for strict volume control in CKD patients.

Properdin Pattern Recognition on Proximal Tubular Cells Is Heparan Sulfate/Syndecan-1 but Not C3b Dependent and Can Be Blocked by Tick Protein Salp20.

Introduction: Proteinuria contributes to progression of renal damage, partly by complement activation on proximal tubular epithelial cells. By pattern recognition, properdin has shown to bind to heparan sulfate proteoglycans on tubular epithelium and can initiate the alternative complement pathway (AP). Properdin however, also binds to C3b(Bb) and properdin binding to tubular cells might be influenced by the presence of C3b(Bb) on tubular cells and/or by variability in properdin proteins in vitro. In this study we carefully evaluated the specificity of the properdin - heparan sulfate interaction and whether this interaction could be exploited in order to block alternative complement activation. Methods: Binding of various properdin preparations to proximal tubular epithelial cells (PTEC) and subsequent AP activation was determined in the presence or absence of C3 inhibitor Compstatin and properdin inhibitor Salp20. Heparan sulfate proteoglycan dependency of the pattern recognition of properdin was evaluated on PTEC knocked down for syndecan-1 by shRNA technology. Solid phase binding assays were used to evaluate the effectivity of heparin(oids) and recombinant Salp20 to block the pattern recognition of properdin. Results: Binding of serum-derived and recombinant properdin preparations to PTECs could be dose-dependently inhibited (P < 0.01) and competed off (P < 0.01) by recombinant Salp20 (IC50: ~125 ng/ml) but not by Compstatin. Subsequent properdin-mediated AP activation on PTECs could be inhibited by Compstatin (P < 0.01) and blocked by recombinant Salp20 (P < 0.05). Syndecan-1 deficiency in PTECs resulted in a ~75% reduction of properdin binding (P = 0.057). In solid-phase binding assays, properdin binding to C3b could be dose-dependently inhibited by recombinant Salp20> heparin(oid) > C3b. Discussion: In this study we showed that all properdin preparations recognize heparan sulfate/syndecan-1 on PTECs with and without Compstatin C3 blocking conditions. In contrast to Compstatin, recombinant Salp20 prevents heparan sulfate pattern recognition by properdin on PTECs. Both complement inhibitors prevented properdin-mediated C3 activation. Binding of properdin to C3b could also be blocked by heparin(oids) and recombinant Salp20. This work indicates that properdin serves as a docking station for AP activation on PTECs and a Salp20 analog or heparinoids may be viable inhibitors in properdin mediated AP activation.

Effect of CDT6 on factors of angiogenic balance in tumour cell lines.

BACKGROUND: Cornea-derived transcript 6 (CDT6, also known as AngX) is an angiopoietin-related factor resulting in anti-tumour effect in vivo. However, a recent abstract reported that CDT6 can also induce angiogenesis and promotes tumour growth. In our previous work, CDT6 had failed to show pro- or anti-angiogenic effects. It is unknown if CDT6 expression occurs in human cancer. MATERIALS AND METHODS: An array of human tumour cell lines and tumour tissues was tested for CDT6-gene expression using RT-PCR. To address the controversial role of CDT6 on angiogenesis in different tumour models, the expression levels of four factors of the angiogenic balance (VEGF, endostatin, TIMP-1 and PAI-1) were determined in CDT6-transfected and control cells of the human and murine melanoma cell lines BLM and B16-F10. Endostatin was significantly up-regulated by CDT6 expression in the human model and significantly down-regulated in the mouse model. None of 18 cell lines or 23 tumours expressed CDT6. CONCLUSION: This contradictory effect on endostatin expression in human and mouse models may be an explanation for the conflicting results for the effect of CDT6 expression on angiogenesis.

Improvement of in vivo transfer of plasmid DNA in muscle: comparison of electroporation versus ultrasound.

Plasmid-based gene delivery to muscle is a treatment strategy for many diseases with potential advantages above viral-based gene delivery methods, however, with a relative low transfection efficiency. We compared two physical methods - electroporation and ultrasound - that facilitate DNA uptake into cells. Mice (C57Bl/6) were injected intramuscular using plasmid DNA encoding an intracellular protein (p53) followed by electroporation or ultrasound. Then 48 hr after the injections the mice were sacrificed. The parameter for transfection efficiency was the area of muscle expressing the transgene. The p53 expression plasmid showed a 36-fold increase (p = 0.015) in transfection efficiency with electroporation compared to ultrasound. Compared with ultrasound, electroporation significantly improves transfection efficiency of naked plasmid DNA transfer into skeletal muscle.

Circulating vascular endothelial growth factor during the normal menstrual cycle.

BACKGROUND: The purpose of the study was to investigate whether cycle-related variations in circulating Vascular Endothelial Growth Factor (VEGF) levels would increase the metastatic potential at specific times during the menstrual cycle. MATERIALS AND METHODS: VEGF levels in serum and whole blood were evaluated during the normal menstrual cycle in premenopausal women. Determination of the menstrual phase was based on hormonal measurements. RESULTS: A total of 46 samples were taken of six menstrual cycles. Serum VEGF was inversely related with progesterone levels (r = -0.6, p = 0.012). Throughout the menstrual cycle the serum VEGF decreased indicating that the lowest VEGF level occurs during the secretory phase, which is compatible with the inverse relationship between serum progesterone and VEGF. CONCLUSION: These findings, however, do not suggest that individual VEGF levels can direct the optimal timing of surgical intervention in breast cancer.

Urinary vitamin D binding protein: a potential novel marker of renal interstitial inflammation and fibrosis.

Non-invasive tubulointerstitial damage markers may allow better titration and monitoring of renoprotective therapy. We investigated the value of urinary vitamin D binding protein excretion (uVDBP) as a tubulointerstitial inflammation and fibrosis marker in adriamycin rats, and tested whether uVDBP parallels renal damage and responds to therapy intensification in humans. In adriamycin (ADR) rats, uVDBP was strongly elevated vs controls (CON) already 6 wks after nephrosis induction (ADR: 727±674 [mean±SD] vs CON: 9±12 µg/d, p<0.01), i.e. before onset of pre-fibrotic and inflammatory tubulointerstitial damage, and at all following 6-wk time points until end of follow up at 30 wks (ADR: 1403±1026 vs CON: 206±132 µg/d, p<0.01). In multivariate regression analysis, uVDBP was associated with tubulointerstitial macrophage accumulation (standardized beta = 0.47, p = 0.01) and collagen III expression (standardized beta = 0.44, p = 0.02) independently of albuminuria. In humans, uVDBP was increased in 100 microalbuminuric subjects (44±93 µg/d) and in 47 CKD patients with overt proteinuria (9.2±13.0 mg/d) compared to 100 normoalbuminuric subjects (12±12 µg/d, p<0.001). In CKD patients, uVDBP responded to intensification of renoprotective therapy (ACEi+liberal sodium: 9.2±13.0 mg/d vs dual RAAS blockade+low sodium: 2747±4013, p<0.001), but remained still >100-fold increased during maximal therapy vs normoalbuminurics (p<0.001), consistent with persisting tubulointerstitial damage. UVDBP was associated with tubular and inflammatory damage markers KIM-1 (standardized beta = 0.52, p<0.001), beta-2-microglobuline (st.beta = 0.45, p<0.001), cystatin C (st.beta = 0.40, p<0.001), MCP-1 (st.beta = 0.31, p<0.001) and NGAL (st.beta = 0.20, p = 0.005), independently of albuminuria. UVDBP may be a novel urinary biomarker of tubulointerstitial damage. Prospectively designed studies are required to validate our findings and confirm its relevance in the clinical setting.

Pro- and anti-apoptotic effects of p53 in cisplatin-treated human testicular cancer are cell context-dependent.

In murine testicular cancer (TC) cells wild-type p53 contributes to sensitivity to DNA-damaging drugs in a dose-dependent way. In human TC, however, the role of wild-type p53 functionality in chemotherapeutic response remains elusive. We analyzed functionality of wild-type p53 in cisplatin sensitivity in the human TC setting using a p53 short interfering (si)RNA approach. The cisplatin-sensitive TC cell line (Tera), the subline with acquired cisplatin resistance (Tera-CP) and a panel of intrinsically resistant TC cell lines (Scha and 2102EP), all expressing wild-type p53, were used. p53 and p53 transcriptional targets MDM2 and p21 (Waf1/Cip1) (p21) were expressed in a p53 transactivation-dependent way in all TC cell lines. Following cisplatin exposure, expression levels of p53 increased, with a subsequent increase in MDM2 and p21 mRNA and protein levels and Fas cell membrane levels. Downregulation of p53 with siRNA lowered cisplatin-induced apoptosis in Tera and Tera-CP, which was associated with a diminished Fas membrane expression. In contrast, p53 suppression augmented cisplatin-induced apoptosis in Scha and 2102EP and concomitantly strongly suppressed MDM2 and p21 mRNA and protein expression. Our results indicate that p53 is involved in transactivation of pro- and anti-apoptotic genes in untreated and cisplatin-treated TC cells, but subtle differences are present between TC cell lines. The opposite role of p53 in cisplatin-induced apoptosis among TC cell lines demonstrates the importance of the cellular context for the p53 transactivation phenotype in TC cells.

Construction of a triple modified p53 containing DNA vaccine to enhance processing and presentation of the p53 antigen.

More effective and less toxic treatments are urgently needed in the treatment of patients with cancer. The tumour suppressor protein p53 is a tumour-associated antigen that could serve that purpose when applied in an immunologic approval to cancer. It is mutated in approximately 50% of the tumours resulting in p53 overexpression, which can serve as target for therapy. To improve the immunisation results in patients with p53 overexpression tumours we constructed a DNA vaccine that could lead to improved processing and presentation of p53 peptides in the MHC-class I. We constructed a triple modified p53 fusion protein containing DNA vaccine by (1) addition of a xeno-antigen (mouse or rat p53 fragment), (2) potentiation of intra-cytoplasmatic accumulation of p53 by deleting the nuclear signalling part, (3) improving the processing to peptides of p53 by addition of ubiquitin. In-vitro experiments confirmed correct construction of the DNA vaccine. Preliminary testing in normal and HLA-A2 mice of this triple modified p53 containing DNA construct meant for human application showed a trend towards a superior immunogenicity.

Platelets and granulocytes, in particular the neutrophils, form important compartments for circulating vascular endothelial growth factor.

The measurement of circulating vascular endothelial growth factor (VEGF) levels as a prognostic factor will gain increasing relevance in the diagnosis and evaluation of treatment in cancer patients. Angiogenesis is an absolute requirement in tumour growth and metastatic disease. In the present study data are presented which indicate that circulating VEGF mainly resides in peripheral blood cells. In 15 healthy volunteers we demonstrated that approximately 34% of the circulating VEGF resides in platelets and approximately 11% in patients with cancer ( n = 4). An important part namely 58% in healthy volunteers and 69% in patients with cancer of the total circulating VEGF is contained in granulocytes, particular in the neutrophils, as confirmed by fluorescence-activated cell sorting (FACS). Also an increased VEGF level per granulocyte is found in patients with cancer (77 microg VEGF/l) compared with the healthy volunteers (164 microg VEGF/l). In contrast only 2% was present in plasma. The biological significance of platelet- or granulocyte-derived VEGF is not yet known. Liberation of VEGF from these compartments could well be of importance for tumour angiogenesis. Therefore, future studies on the clinical value of circulating VEGF as a prognostic factor in cancer patients should include measurements of VEGF in peripheral blood cells.