Humoral Response to Hepatitis B and COVID-19 Vaccine among Maintenance Hemodialysis Patients.

Maintenance hemodialysis (MHD) patients have impaired immunological responses to pathogens and vaccines. In this study, we compared the humoral response to HBV and COVID-19 vaccines in a cohort of MHD patients. Demographic and clinical characteristics of vaccine responders and non-responders were also compared, and the association between the humoral responses to both vaccines was evaluated. The cohort included 94 MHD patients who were vaccinated at least once for HBV and twice for COVID-19. Among the 94 patients, 28 (29.8%) did not develop protective titers to HBV. Hypertension, coronary heart disease, and heart failure were more common in non-responders. Among MHD patients, 85% had positive IgG anti-spike SARS-CoV-2 levels 6 months after two doses of BNT162b2 (Pfizer/Biotech) vaccine. Age and immunosuppressive therapy were the main predictors of humoral response to COVID-19 vaccine. We did not find any association between non-responders to HBV and non-responders to COVID-19 vaccine. There was no difference in IgG anti-spike titers between HBV responders and non-responders (505 ± 644 vs. 504 ± 781, p = 0.9) Our results suggest that reduced humoral response to hepatitis B is not associated with reduced response to COVID-19 vaccine. Different risk-factors were associated with poor immune response to HBV and to COVID-19 vaccines.

Early effectiveness of BNT162b2 Covid-19 vaccine in preventing SARS-CoV-2 infection in healthcare personnel in six Israeli hospitals (CoVEHPI).

BACKGROUND: Methodologically rigorous studies on Covid-19 vaccine effectiveness (VE) in preventing SARS-CoV-2 infection are critically needed to inform national and global policy on Covid-19 vaccine use. In Israel, healthcare personnel (HCP) were initially prioritized for Covid-19 vaccination, creating an ideal setting to evaluate early real-world VE in a closely monitored population. METHODS: We conducted a prospective study among HCP in 6 hospitals to estimate the effectiveness of the BNT162b2 mRNA Covid-19 vaccine in preventing SARS-CoV-2 infection. Participants filled out weekly symptom questionnaires, provided weekly nasal specimens, and three serology samples - at enrollment, 30 days and 90 days. We estimated VE against PCR-confirmed SARS-CoV-2 infection using the Cox Proportional Hazards model and against a combined PCR/serology endpoint using Fisher's exact test. RESULTS: Of the 1567 HCP enrolled between December 27, 2020 and February 15, 2021, 1250 previously uninfected participants were included in the primary analysis; 998 (79.8%) were vaccinated with their first dose prior to or at enrollment, all with Pfizer BNT162b2 mRNA vaccine. There were four PCR-positive events among vaccinated participants, and nine among unvaccinated participants. Adjusted two-dose VE against any PCR-confirmed infection was 94.5% (95% CI: 82.6%-98.2%); adjusted two-dose VE against a combined endpoint of PCR and seroconversion for a 60-day follow-up period was 94.5% (95% CI: 63.0%-99.0%). Five PCR-positive samples from study participants were sequenced; all were alpha variant. CONCLUSIONS: Our prospective VE study of HCP in Israel with rigorous weekly surveillance found very high VE for two doses of Pfizer BNT162b2 mRNA vaccine against SARS-CoV-2 infection in recently vaccinated HCP during a period of predominant alpha variant circulation. FUNDING: Clalit Health Services.

Vitamin D diminishes the high platelet aggregation of type 2 diabetes mellitus patients.

Platelet activation is found in inflammatory conditions and implicated in the pathogenesis of chronic medical conditions, such as atherosclerosis, coronary vascular disease, cerebrovascular disease, and diabetes mellitus (DM). HbA1c is inversely related to vitamin D25 levels in individuals with and without DM. This study aimed to determine the relation between platelet aggregation, vitamin D and HbA1c among healthy individuals and those with Type 2 DM (T2DM). The direct effect of vitamin D1, 25 (calcitriol) on platelet aggregation was also investigated. The study included four groups: A. normoglycemic Control group: HbA1c<5.7%; B. Pre-diabetes (DM): 5.7% ≥ HbA1c ≤ 6.4%; C. DM on aspirin therapy: HbA1c>6.4%(+)Asp.; and D. DM not on aspirin therapy: HbA1c > 6.4%(-)Asp. Platelet aggregation was tested with and without calcitriol or saline pre-treatment, using collagen or adenosine diphosphate (ADP) as agonists. Platelet aggregation was higher in DM(-)Asp group compared to normoglycemic and DM(+)Asp, and higher, but not significant compared to pre-DM. The entire study population exhibited negative correlation between HbA1c and serum concentration of vitamin D25. Excluding DM(+)Asp, aggregation induced by collagen was significantly higher in patients with insufficient (<76 nmol/L) vitamin D25 compared to sufficient (≥76 nmol/L) vitamin D25. In this cohort, a negative correlation was found between serum concentrations of vitamin D25 and collagen-induced percent maximum (%max) aggregation and area under curve (AUC) aggregation. In the DM(-)Asp group, collagen-induced aggregation was reduced by approximately 25% after calcitriol treatment. Calcitriol decreased ADP-induced aggregation of control and DM(+)Asp groups to approximately 85% of saline treatment. We conclude that glycemic control is inversely associated with high platelet aggregation and low vitamin D25 levels. This elevated aggregation could be regulated by a novel, direct effect of calcitriol, indicating a beneficial effect of vitamin D on vascular complications related to diabetes. We offer a possible non-genomic mechanism for the vitamin D/Vitamin D receptor (VDR) pathway.

Chemical and thyroid hormone profile of the bone marrow interstitial fluid in hematologic disorders and patients without primary hematologic disorders.

Bone marrow interstitial fluid (BMIF) has not been well characterized. BMIF was isolated from 60 patients including plasma cell dyscrasias (PCD, n = 33), other primary hematologic disorders (OHD, n = 15), and patients with secondary or nonhemtologic disorders (NHD, n = 12) and analyzed for an array of chemical constituents. These included total cholesterol, glucose, phosphate, creatinine, urea, total protein, albumin, globulins, total bilirubin, aspartate aminotransferase, lactate dehydrogenase, sodium, osmolarity, free triiodothyronine (free T3), total triiodothyronine (total T3), and free tetraiodothyronine (free T4). Levels of BMIF components were compared between patient groups and to plasma levels. Compared with plasma, total cholesterol, total protein, total bilirubin, sodium, and calculated osmolarity were lower in BMIF in all groups (P < 0.05). Calculated globulins and aspartate aminotransferase were lower in BMIF of PCD patients and patients with NHD. Albumin was lower in BMIF of patients with PCD and patients with OHD. Lastly, free T4 was significantly higher in BMIF of patients with PCD and patients with OHD. Similar results were demonstrated in a separate analysis performed in patients with multiple myeloma. To conclude, the chemical and thyroid hormone composition of BMIF differs significantly from plasma in several key constituents.

[THE EFFECT OF VITAMIN D ON THE EXPRESSION OF ADAMTS13 IN CULTURED ENDOTHELIAL CELLS EXPOSED TO A DIABETIC-LIKE ENVIRONMENT].

BACKGROUND: Diabetes mellitus (DM) is the leading cause of end-stage kidney disease. Inflammation, fibrosis, coagulability and oxidative stress exacerbate kidney disease. The glycoprotein, Von Willebrand Factor (VWF) has a crucial role in platelet thrombus formation. The enzyme ADAMTS13 is responsible for VWF cleavage. Both are important in the interface between diabetic nephropathy, hypercoagulability and atherosclerotic cardiovascular disease. Vitamin D inhibits endothelial proliferation, blunts angiogenesis and is a cardioprotective agent. This study evaluated the role of vitamin D on ADAMTS13 activity in human umbilical vein endothelial cells (HUVEC) exposed to a diabetic-like environment. METHODS: HUVEC were stimulated with 200µg/µl AGE-HSA, 250mg/dl glucose, and 10-10mol/l vitamin D for 24 hours. Western blot and ELISA techniques were used to determine ADAMTS13 protein expression and IL6 and IL8 protein secretion. RESULTS: ADAMTS13 protein expression decreased and IL6 and IL8 protein secretion increased in HUVEC exposed to a diabetic-like environment. The addition of vitamin D significantly down-regulated IL6 and IL8 secretion and up-regulated ADAMST13 expression. CONCLUSIONS: Decreased ADAMTS13 expression in HUVEC exposed to a diabetic-like environment may contribute to the pathogenesis of hypercoagulability observed in DM. Normalization of ADAMTS13 by vitamin D may contribute to improvement in hypercoagulability.

Effect of Vitamin D Status on Von Willebrand Factor and ADAMTS13 in Diabetic Patients on Chronic Hemodialysis.

Von Willebrand factor (vWF) is a glycoprotein with a crucial role in the formation of platelet thrombi, and ADAMTS13 is the main enzyme responsible for vWF cleavage. Both are important in the relationship between diabetic nephropathy, hypercoagulability, and cardiovascular disease. This study evaluated a potential relationship between vitamin D (vitD) levels, vWF, ADAMTS13 activity, and inflammation in diabetic patients on chronic hemodialysis (HD). Blood samples from 52 diabetic patients on chronic HD were obtained to determine vitD levels, vWF, and ADAMTS13 activity, and inflammatory markers. HD patients were grouped according to 25-hydroxyvitamin D [25(OH) VitD]<25 nmol/L (n=16) or >25 nmol/L (n=36). vWF antigen and vWF activity were elevated in both groups, with an average of 214.3±82.6% and 175.8±72.6%, respectively. Average ADAMTS13 activity was within the normal range in both groups. Blood samples from the vitD <25 nmol/L group showed a positive correlation between c-reactive protein (CRP) and vWF levels (P=0.023; r=0.564; 95% confidence interval=0.095-0.828), with a negative correlation between HbA1c and 25(OH) VitD (P=0.015; r=-0.337; 95% confidence interval=-0.337-0.19). Diabetic patients on chronic HD had elevated vWF levels and activity with no significant change in ADAMTS13 activity. The correlation between CRP and vWF levels in the 25(OH) VitD<25 nmol/L group suggests inflammatory-related endothelial dysfunction in these patients.

[REPORTING CRITICAL LAB RESULTS, A CHALLENGE FOR THE LAB AND THE PHYSICIAN - A SUMMARY OF FOUR YEARS OF EXPERIENCE IN MEIR MEDICAL CENTER LABORATORIES].

INTRODUCTION: Critical laboratory results require prompt reporting to the attending physician, as they may indicate that a patient is in a life-threatening condition. Although this important subject has been covered in many publications, it needs more attention from our healthcare organizations, which have no official policy on the subject. Matching expectations between the doctor and the laboratory needs to be better defined. PURPOSE: The aim of this work was to inform the community of doctors and laboratories about the multiple problems concerning the reporting of critical laboratory results, to create a platform for exchanging views and ideas, and to build an extensive infrastructure for developing a unified plan to address this important issue. METHODS: We present the results of four years of experience of reporting critical laboratory values at the Meir Medical Center Laboratories. The idea leading this work was to present the relatively low rate of critical results reported by the laboratories in 2010, sharing the problems discovered while investigating the situation in depth, and presenting the solutions that enabled us to obtain the desired results within four years. RESULTS: Gradual implementation of these improvements resulted in critical value reporting increasing from 55% in 2010 to 95% currently. CONCLUSION: We suggest a model for improving critical laboratory values reporting based on our 4-year experience, which emphasizes: (1) The importance of selecting proper tests and values for critical results; (2) The significance of using technology and computerized measures to support the process; and (3) Developing quick procedures for monitoring and controlling the process.

Bevacizumab attenuates major signaling cascades and eIF4E translation initiation factor in multiple myeloma cells.

Multiple myeloma (MM), a malignancy of plasma cells, remains fatal despite introduction of novel therapies, partially due to humoral factors, including vascular endothelial growth factor (VEGF), in their microenvironment. The aim of this study was to explore the efficacy of anti-VEGF treatment with bevacizumab directly on MM cells. Particular attention was directed to the affect of VEGF inhibition on protein translation initiation. Experiments were conducted on MM cells (lines, bone marrow (BM) samples) cultured on plastic. Inhibition of VEGF was achieved with the clinically employed anti-VEGF antibody, bevacizumab, as a platform and its consequences on viability, proliferation, and survival was assessed. VEGF downstream signals of established importance to MM cell biology were assayed as well, with particular emphasis on translation initiation factor eIF4E. We showed that blocking VEGF is deleterious to the MM cells and causes cytostasis. This was evidenced in MM cell lines, as well as in primary BM samples (BM MM). A common bevacizumab-induced attenuation of critical signaling effectors was determined: VEGFR1, mTOR, c-Myc, Akt, STAT3, (cell lines) and eIF4E translation initiation factor (lines and BM). ERK1/2 displayed a variegated response to bevacizumab (lines). Utilizing a constitutively Akt-expressing MM model, we showed that the effect of bevacizumab on viability and eIF4E status is Akt-dependent. Of note, the effect of bevacizumab was achieved with high concentrations (2 mg/ml), but was shown to be specific. These findings demonstrate that bevacizumab has a direct influence on major pathways critically activated in MM that is independent from its established effect on angiogenesis. The cytostatic effect of VEGF inhibition on MM cells underscores its potential in combined therapy, and our findings, regarding its influence on translation initiation, suggest that drugs that unbalance cellular proteostasis may be particularly effective.

Pleural ELFA D-dimer assay: a surrogate marker for malignant pleural effusion.

BACKGROUND: Malignant pleural effusion is associated with enhanced fibrinolysis. However, no data are available concerning the precise role of pleural D-dimer assay in pleural effusion. We therefore assessed the role of pleural D-dimer assay in predicting malignant pleural effusion. PATIENTS AND METHODS: A prospective laboratory investigation was conducted in a tertiary care teaching hospital. The study included consecutive patients with pleural effusion who presented at the Pulmonary Department between November 2009 and May 2010. Blood and pleural D-dimer levels were measured by Enzyme Linked Fluorescent assay (ELFA). The results were correlated with the clinical, laboratory, and radiological findings, and with the final diagnosis of the pleural fluid. RESULTS: A total of 103 patients with pleural effusion were included in the study. The Pleural ELFA D-dimer results were found to be positively correlated with pleural etiology of malignancy (p=0.0001). Pleural etiology was also correlated with pleural LDH, pleural protein, pleural PH, pleural glucose, pleural and blood CRP, but not with ADA. In a binary logistic regression, only the pleural ELFA D-dimer assay was a significant predictor of the malignant pleural effusion (odds ratio 1.007; 95% confidence interval 1.002-1.012; p=0.007). The area under the receiver operating characteristics curve for malignancy was 0.79. A D-dimer level of 146mg/ml had a sensitivity of 82% and a specificity of 74%. CONCLUSIONS: We found high D-dimer levels among malignant pleural effusion. D-dimer might be useful as a simple, noninvasive, surrogate marker for malignant pleural effusion.

Indole-3-carbinol inhibits telomerase activity and gene expression in prostate cancer cell lines.

BACKGROUND: Indole-3-carbinol (I3C) is a phytochemical with anticarcinogenic properties. Telomerase activity is key in carcinogenesis. We investigated the effect of I3C on telomerase in human prostate cancer cell lines LNCaP and PC3. MATERIALS AND METHODS: Cells were treated with I3C at 100 and 250 µM with and without 10-50 µM diethylstilbestrol (DES). Telomerase activity was performed using TRAPaze Telomerase Detection Kit, and hTERT gene expression by real time quantitative RT-PCR. RESULTS: I3C (250 µM) inhibited telomerase activity and mRNA expression of hTERT in LNCaP and PC3 cells. I3C at 250 µM combined with any concentration of DES was cytotoxic to LNCaP. Telomerase activity in PC3 cells with 250 µM of I3C and 25 or 50 µM of DES was significantly reduced or inhibited, respectively. I3C combined with DES reduced PC3 viability and eliminated LNCaP cells. CONCLUSION: I3C significantly inhibited telomerase activity and hTERT mRNA expression in LNCaP and PC3 cells. Combination of I3C and DES enhanced the inhibitory effect on telomerase activity, gene expression, and cell viability. These results implied that I3C and DES combined might help in prostate cancer treatment.

The synthetic estrogen diethylstilbestrol (DES) inhibits the telomerase activity and gene expression of prostate cancer cells.

BACKGROUND: Telomerase, which lengthens telomeres, is normally down-regulated in somatic cells and highly up-regulated in dividing cells, such as malignant cells. Human prostate cancer is androgen dependent. Estrogens, including the synthetic estrogen diethylstilbestrol (DES), are used in prostate cancer treatment to reduce androgen levels via feedback inhibition of the hypothalamic release of luteinizing hormone releasing hormone (LH-RH). DES has also direct anticancer activities, such as apoptosis induction. We investigated in vitro the effect of DES on telomerase activity and on gene expression in the presence and absence of androgens. We used two prostate cancer cell lines: LNCaP (androgen dependent) and PC3 (androgen independent). METHODS: LNCaP and PC3 cells were treated with 0.1-1,000 nM testosterone or dihydrotestosterone (DHT) in the presence of DES (25 or 50 microM). Cell telomerase activity and gene expression (mRNA) were measured. RESULTS: LNCaP: As expected, testosterone and DHT significantly increased telomerase activity and gene expression. However, these effects were inhibited by DES. Contrary to expectations, the combination of DES and testosterone functioned synergistically leading to complete inhibition of telomerase activity. PC3: Testosterone and DHT did not affect telomerase activity and gene expression, whereas DES, in the absence or presence of the androgens, significantly inhibited telomerase activity. CONCLUSIONS: In the present study, we demonstrated the ability of DES to inhibit telomerase in prostate cancer cells. Androgens did not limit the inhibitory effect and even acted synergistically with DES in the LNCaP line. This phenomenon should be considered if telomerase inhibition is part of prostate cancer treatment.

Low extracellular Ca2+: a mediator of endothelial inflammation.

BACKGROUND: Recent studies have suggested that vitamin D and an imbalance in calcium homeostasis may have an impact on the cardiovascular system. The aim of this study was to assess the impact of different concentrations of extracellular Ca(2+) on human umbilical vein cord endothelial cells (HUVEC) by measuring its effect on parameters involved in the pathogenesis of vascular inflammatory responses. METHODS: HUVEC were grown in the 3.5, 4.5 or 5.8 mg/dL concentration of extracellular Ca(2+) for 2-3 weeks. Expression of adhesion molecules was analysed by flow cytometry. Endothelial nitric oxide synthase (eNOS), receptor of advanced glycation end-product (RAGE) and interleukin-6 (IL-6) mRNA expressions were determined by real-time PCR. eNOS, inhibitor kappa Balpha (IkappaBalpha) and phosphorylated IkappaBalpha protein levels by Western blot, eNOS activity by conversion of [(14)C]-arginine to [(14)C]-citrulline, IL-6 and osteoprotegerin (OPG) secretion by ELISA and DNA-binding activity of nuclear factor kappa B (NFkappaB)-p65 were assayed colorimetrically in nuclear extracts. RESULTS: In the presence of low Ca(2+) (3.5 mg/dL), protein expressions and activity of eNOS were diminished, while the protein expressions of intercellular adhesion molecule-1 (ICAM-1), as well as the mRNA expressions of RAGE and IL-6, were elevated. The protein secretions of IL-6 and OPG were also stimulated in low Ca(2+) concentration. At this concentration, the DNA-binding activity of NFkappaB was enhanced, probably due to the decreased level of IkappaBalpha. CONCLUSIONS: These results suggest that lower extracellular ionized Ca(2+) may play a relevant role in modifying endothelial cells functions.

Parathyroid hormone decreases endothelial osteoprotegerin secretion: role of protein kinase A and C.

Parathyroid hormone (PTH), which is elevated in patients with chronic renal failure, has been shown to participate in the development of vascular calcification. Previous studies have demonstrated that PTH may promote endothelial expressions of proinflammatory parameters. On the basis of these data, we evaluated whether PTH may have an impact on endothelial osteoprotegerin (OPG), a vascular-protective factor which may control vascular calcification. Endothelial cells were stimulated with 10(-12) to 10(-10) mol/l PTH. PKC and PKA are the main cellular pathways of PTH. Inhibitors and activators of PKC or PKA were used to determine whether these signaling pathways are involved in the control of endothelial OPG. PTH induced a decrease in OPG secretion and mRNA expression. Treatment of PTH-stimulated cells by calphostin C (PKC inhibitor) induced a further decrease in OPG secretion, while Rp-cAMP (PKA inhibitor) had no additional effect. In nonstimulated cells, a PKC activator significantly stimulated OPG secretion, while a PKA activator was associated with a decline. These effects were blunted in the presence of calphostin C and Rp-cAMP, respectively. An increase in OPG secretion induced by a PKC activator indicates that the basal OPG secretion is mediated through PKC. The decrease induced by a PKA activator, which is similar to the decrease observed with PTH, suggests that the action of PTH on OPG secretion and mRNA expression may be due to the PKA pathway.

Calcitriol blunts the deleterious impact of advanced glycation end products on endothelial cells.

Advanced glycation end products (AGEs), which are elevated in diabetic and uremic patients, may induce vascular dysfunctions, and calcitriol may improve the cardiovascular complications. Therefore, we examined whether calcitriol may modify the endothelial response to AGEs stimulation. Knowing the importance of nuclear factor-kappaB in endothelial inflammatory responses, the effect of AGEs and calcitriol on this pathway was also studied. Calcitriol was added to endothelial cells previously incubated with AGE-human serum albumin (HSA). AGE-HSA induced a decrease in endothelial nitric oxide synthase (eNOS) mRNA expression and enzyme activity. Addition of calcitriol to AGE-HSA-treated endothelial cells improved the decreased action of AGEs on the eNOS system. AGE-HSA increased the AGEs receptor mRNA and protein, which were both blunted by calcitriol. The parallel elevation of interleukin-6 mRNA in the presence of AGE-HSA was also blunted by calcitriol. The NF-kappaB-p65 DNA binding activity was enhanced and associated with a decrease in inhibitor kappaBalpha (IkappaBalpha) and an increase in phosphorylated (p)-IkappaBalpha levels. Addition of calcitriol blunted the AGEs-induced elevation of NF-kappaB-p65 DNA binding activity, a phenomenon related to an increased expression of IkappaBalpha. This increase was correlated to declined p-IkappaBalpha levels. The present results support the concept that calcitriol may act as a vascular protective agent counteracting the probable deleterious actions of AGEs on endothelial cell activities.

Parathyroid hormone stimulates the endothelial nitric oxide synthase through protein kinase A and C pathways.

BACKGROUND: Parathyroid hormone (PTH), the major systemic calcium regulating hormone has been implicated in the development of hypertension and the occurrence of uraemic vascular changes. As nitric oxide synthase (NOS) is involved in the production of nitric oxide, and acute PTH effect is characterized by vasodilation, the effect of PTH on the endothelial NOS (eNOS) system was measured in cultured human umbilical cord vein endothelial cells (HUVEC) and the pathways possibly involved were studied. METHODS: The presence of the PTH receptor-1 (PTHR1) on the HUVEC membrane was examined by RT-PCR, immunocytochemistry and western blot. HUVEC were stimulated with 10(-12) to 10(-10) mol/l PTH. The eNOS mRNA expression was established by RT-PCR and the eNOS protein levels were determined by western blot. The eNOS activity was measured by the conversion of [(14)C]arginine to [(14)C]citrulline. RESULTS: PTHR1 has been found to be expressed in HUVEC and its expression is depressed by increasing concentrations of PTH. PTH induced a significant increase in eNOS mRNA (10(-11) mol/l: 1.87 +/- 0.16, P = 0.012; 10(-10) mol/l: 1.96 +/- 0.28, P = 0.007, fold of control), and protein expression. The eNOS activity was also significantly stimulated (10(-11) mol/l: 1139 +/- 203; 10(-10) mol/l: 1323 +/- 216 vs control: 621 +/- 154 cpm/150 mug protein, P < 0.01). The addition of calphostin C (PKC inhibitor) or Rp-cAMP (PKA inhibitor) reduced the eNOS mRNA, protein expression and activity of PTH-stimulated HUVEC. The combined treatment of calphostin C and Rp-cAMP abolished the eNOS protein expression and activity. CONCLUSION: PTH induces an increased activity of the eNOS system; probably both PKA and PKC pathways are involved in this activation. Such data may explain the vasodilation observed after acute treatment with PTH.

Additive renoprotective effect of candesartan and tetrahydrobiopterin in rats after 5/6 nephrectomy.

BACKGROUND: Chronic treatment with candesartan cilexetil (C) improves the outcome of rats after 5/6 nephrectomy (Nx). Tetrahydrobiopterin (BH4), an essential cofactor for appropriate endothelial nitric oxide synthase (eNOS) activity, prevents an increase in blood pressure (BP) in Nx rats when given immediately after surgery. In the present study, we evaluated the renoprotective effect of a combined treatment. METHODS: Five groups of rats were studied: SHAM (sham-operated rats, n=12); SNx (untreated 5/6 nephrectomized rats, n=15); C (SNx rats treated with candesartan cilexetil, 5 mg/kg/day per os, n=11); C+BH4 (SNx rats treated with candesartan cilexetil and BH4, 10 mg/kg/day intraperitoneally, n=11); and BH4 (SNx rats treated with BH4, 10 mg/kg/day intraperitoneally, n=11). Treatment began 30 days after surgery, when hypertension and renal insufficiency have developed. This day was considered as day 1 of treatment for statistical comparisons. The study was continued until 50% mortality was achieved in the SNx rats (4 months after surgery). RESULTS: The survival rates were 100% for SHAM, 47% for SNx, 50% for BH4, 64% for C and 80% for C+BH4 (P<0.05 vs all). Untreated Nx rats developed hypertension, proteinuria (UP) and severe renal insufficiency. Mortality was associated with a lower renal function and increased urine protein excretion. In C and C+BH4 rats, systolic blood pressure (SBP) decreased significantly. BH4 alone had a mild non-significant effect on SBP. C and C+BH4 treatments attenuated significantly the increase in proteinuria found in SNx animals. The weight of the remnant kidneys as well as the severity of glomerulosclerosis were significantly lower in the C+BH4 rats. CONCLUSION: This study shows that in subnephrectomized rats, addition of BH4 to a treatment with candesartan had an additive renoprotective effect. The mechanism of such action may include a better control of BP associated with a blockade of actions of angiotensin II (Ag II), an improvement in nitric oxide synthesis and a balanced redox.

Halofuginone reduces the occurrence of renal fibrosis in 5/6 nephrectomized rats.

BACKGROUND: Halofuginone is a novel antifibrotic agent that can reverse the fibrotic process by specific inhibition of collagen type I synthesis. OBJECTIVES: To evaluate the effect of Halo on the development of glomerulosclerosis and interstitial fibrosis in the 5/6 nephrectomy rat model METHODS: Male Wistar rats were assigned to undergo 5/6 NX or sham operation, and then divided into three groups: 5/6 NX rats (NX-Halo and NX-Control) and sham. Systolic blood pressure, proteinuria and body weight were determined every 2 weeks. At sacrifice (10 weeks) creatinine clearance was evaluated and remnant kidneys removed for histologic examination, sirius red staining and in situ hybridization RESULTS: Systolic blood pressure increased progressively in both 5/6 NX groups. Halo slowed the increase in proteinuria in 5/6 NX rats. As expected, creatinine clearance was lower in 5/6 NX groups when compared to sham rats. Creatinine clearance was significantly higher in the NX-Halo group at the end of the study period. Histologic examination by light microscopy showed significantly less severe interstitial fibrosis and glomerulosclerosis in Halo-treated rats. The increase in collagen alpha1 (I) gene expression and collagen staining after nephrectomy was almost completely abolished by Halo. CONCLUSIONS: Halofuginone reduced proteinuria as well as the severity of interstitial fibrosis and glomerulosclerosis in 5/6 NX rats. The renal beneficial effect of Halo was also demonstrated by the blunted decrease in creatinine clearance observed in the treated animals.

Parathyroid hormone stimulates endothelial expression of atherosclerotic parameters through protein kinase pathways.

Parathyroid hormone (PTH), the major systemic calcium-regulating hormone, has been linked to uremic vascular changes. Considering the possible deleterious action of PTH on vascular structures, it seemed logical to evaluate the impact of PTH on the receptor of advanced glycation end products (RAGE) and interleukin 6 (IL-6) mRNA and protein expression, taking into account that such parameters might be involved in the pathogenesis of vascular calcification, atherosclerosis, and/or arteriolosclerosis. Human umbilical vein cord endothelial cells (HUVEC) were stimulated for 24 h with 10(-12)-10(-10) mol/l PTH. The mRNA expression of RAGE and IL-6 was established by reverse transcriptase/PCR techniques. RAGE protein levels were determined by Western blot and IL-6 secretion was measured by ELISA. The pathways by which PTH may have an effect on HUVEC functions were evaluated. PTH (10(-11)-10(-10)mol/l) significantly increased RAGE mRNA and protein expression. PTH also significantly increased IL-6 mRNA expression without changes at protein levels. The addition of protein kinase (PKC or PKA) inhibitors or nitric oxide (NO) synthase inhibitors significantly reduced the RAGE and IL-6 mRNA expression and the RAGE protein expression. PTH stimulates the mRNA expressions of RAGE and IL-6 and the protein expression of RAGE. These stimulatory effects are probably through PKC and PKA pathways and are also NO dependent. Such data may explain the possible impact of PTH on the atherosclerotic and arteriosclerotic progression.

Advanced glycation end products stimulate tumor necrosis factor-alpha and interleukin-1 beta secretion by peritoneal macrophages in patients on continuous ambulatory peritoneal dialysis.

BACKGROUND: Advanced glycation end products, formed by the non-enzymatic glycation of proteins with reducing sugars, are thought to play a pathogenetic role in the vascular complications of diabetes, uremia and atherosclerosis. 132-microglobulin is a major constituent of amyloid fibrils in dialysis-related amyloidosis. AGE1-modified beta2m has been found in amyloid deposits of long-term hemodialysis patients. AGE-modified 132m has also been shown to enhance chemotaxis and increase tumor necrosis factor-alpha and interleukin-1 beta secretion by circulating and tissue monocytes/macrophages. OBJECTIVES: To investigate the effect of AGE-modified 132m and AGE-human serum albumin on TNF-alpha and IL-1beta secretion by human peritoneal macrophages derived from patients on continuous ambulatory peritoneal dialysis. METHODS: Human PMO were isolated from peritoneal dialysis effluent of stable CAPD patients and were incubated for 24 hours with AGE-modified beta2m, beta2m, AGE-HSA, HSA or lipopolysaccharide. TNF-alpha or IL-1beta secretion was measured by enzyme-linked immunosorbent assay in cell-free culture supernatants. RESULTS: Both AGE-modified beta2m and AGE-HSA significantly increased TNF-alpha and IL-1beta secretion by human PMO in a dose-dependent manner (50-200 microg/ml). In contrast, beta2m or HSA had no such stimulatory effect on TNF-alpha secretion but had a small significant increase in IL-1beta secretion. CONCLUSIONS: AGE-modified beta2m promotes in vitro TNF-alpha and IL-13 secretion by human PMO of CAPD patients. Activation of these macrophages by AGE-modified beta2m may be a contributory factor to the morphologic changes and altered permeability of the peritoneal membrane in long-term CAPD.

Effect of advanced glycation end-products on gene expression and synthesis of TNF-alpha and endothelial nitric oxide synthase by endothelial cells.

BACKGROUND: Advanced glycation end-products (AGEs), which are formed in aging, diabetes mellitus, and kidney failure are implicated in the occurrence of vascular complications. We, thus, evaluated in cultured endothelial cells, the AGEs' effect on gene expression and synthesis of tumor necrosis factor-alpha (TNF-alpha) and endothelial nitric oxide synthase (eNOS) mRNA and protein expression, which may be involved in vascular remodeling. METHODS: Human umbilical vein cords endothelial cells (HUVEC) were stimulated with AGE-specific compounds [AGE-human serum albumin (AGE-HSA), N(epsilon)-carboxymethylysine (CML), AGE-beta2 microglobulin (AGE-beta2m)], and thereafter, incubated with interleukin1-alpha, lipopolysaccharide, and interferon-gamma. RESULTS: mRNA expression and secretion of TNF-alpha were significantly enhanced after incubation with AGE-HSA, CML, and AGE-beta2m compared to that found in HUVEC incubated with HSA or beta2m. AGE-HSA, CML, and AGE-beta2m induced a significant decrease in eNOS protein and mRNA expression. CONCLUSION: AGEs promote mRNA expression and secretion of TNF-alpha and reduce eNOS mRNA and protein expression in HUVEC. Such changes may play a role in the vascular dysfunction and the development of vasculopathy seen in diabetes, uremia, and old age.