Low serum 1,25(OH)2D3 in end-stage renal disease: is reduced 1α-hydroxylase the only problem?

Low serum 1,25-dihydroxyvitamin D (1,25(OH)2D) in end-stage renal disease (ESRD) is considered a consequence of elevated fibroblast growth factor 23 (FGF23) and concomitant reduced activity of renal 1α-hydroxylase (CYP27B1). Current ESRD treatment strategies to increase serum calcium and suppress secondary hyperparathyroidism involve supplementation with vitamin D analogues that circumvent 1α-hydroxylase. This overlooks the potential importance of 25-hydroxyvitamin D (25(OH)D) deficiency as a contributor to low serum 1,25(OH)2D. We investigated the effects of vitamin D (cholecalciferol) supplementation (40,000 IU for 12 weeks and maintenance dose of 20,000 IU fortnightly), on multiple serum vitamin D metabolites (25(OH)D, 1,25(OH)2D3 and 24,25(OH)2D3) in 55 haemodialysis patients. Baseline and 12 month data were compared using related-samples Wilcoxon signed rank test. All patients remained on active vitamin D analogues as part of routine ESRD care. 1,25(OH)2D3 levels were low at baseline (normal range: 60-120 pmol/L). Cholecalciferol supplementation normalised both serum 25(OH)D and 1,25(OH)2D3. Median serum 25(OH)D increased from 35.1 nmol/L (IQR: 23.0-47.5 nmol/L) to 119.9 nmol/L (IQR: 99.5-143.3 nmol/L) (P < 0.001). Median serum 1,25(OH)2D3 and 24,25(OH)2D3 increased from 48.3 pmol/L (IQR: 35.9-57.9 pmol/L) and 3.8 nmol/L (IQR: 2.3-6.0 nmol/L) to 96.2 pmol/L (IQR: 77.1-130.6 pmol/L) and 12.3 nmol/L (IQR: 9-16.4 nmol/L), respectively (P < 0.001). A non-significant reduction in daily active vitamin D analogue dose occurred, 0.94 µmcg at baseline to 0.77 µmcg at 12 months (P = 0.73). The ability to synthesise 1,25(OH)2D3 in ESRD is maintained but is substrate dependent, and serum 25(OH)D was a limiting factor at baseline. Therefore, 1,25(OH)2D3 deficiency in ESRD is partly a consequence of 25(OH)D deficiency, rather than solely due to reduced 1α-hydroxylase activity as suggested by current treatment strategies.

Impaired arterial vitamin D signaling occurs in the development of vascular calcification.

Conflicting data exists as to whether vitamin D receptor agonists (VDRa) are protective of arterial calcification. Confounding this, is the inherent physiological differences between human and animal experimental models and our current fragmented understanding of arterial vitamin D metabolism, their alterations in disease states and responses to VDRa's. Herein, the study aims to address these problems by leveraging frontiers in human arterial organ culture models. Human arteries were collected from a total of 24 patients (healthy controls, n = 12; end-stage CKD, n = 12). Cross-sectional and interventional studies were performed using arterial organ cultures treated with normal and calcifying (containing 5mmol/L CaCl2 and 5mmol/L ß-glycerophosphate) medium, ex vivo. To assess the role of VDRa therapy, arteries were treated with either calcitriol or paricalcitol. We found that human arteries express a functionally active vitamin D system, including the VDR, 1α-hydroxylase and 24-hydroxylase (24-OHase) components and these were dysregulated in CKD arteries. VDRa therapy increased VDR expression in healthy arteries (p<0.01) but not in CKD arteries. Arterial 1α-OHase (p<0.05) and 24-OHase mRNA and protein expression were modulated differentially in healthy and CKD arteries by VDRa therapy. VDRa exposure suppressed Runx2 and MMP-9 expression in CKD arteries, however only paricalcitol suppressed MMP-2. VDRa exposure did not modulate arterial calcification in all organ culture models. However, VDRa reduced expression of senescence associated ß-galactosidase (SAßG) staining in human aortic-smooth muscle cells under calcifying conditions, in vitro. In conclusion, maladaptation of arterial vitamin D signaling components occurs in CKD. VDRa exposure can exert vasculo-protective effects and seems critical for the regulation of arterial health in CKD.

Cardiovascular Functional Reserve Before and After Kidney Transplant.

Importance: Restitution of kidney function by transplant confers a survival benefit in patients with end-stage renal disease. Investigations of mechanisms involved in improved cardiovascular survival have relied heavily on static measures from echocardiography or cardiac magnetic resonance imaging and have provided conflicting results to date. Objectives: To evaluate cardiovascular functional reserve in patients with end-stage renal disease before and after kidney transplant and to assess functional and morphologic alterations of structural-functional dynamics in this population. Design, Setting, and Participants: This prospective, nonrandomized, single-center, 3-arm, controlled cohort study, the Cardiopulmonary Exercise Testing in Renal Failure and After Kidney Transplantation (CAPER) study, included patients with stage 5 chronic kidney disease (CKD) who underwent kidney transplant (KTR group), patients with stage 5 CKD who were wait-listed and had not undergone transplant (NTWC group), and patients with hypertension only (HTC group) seen at a single center from April 1, 2010, to January 1, 2013. Patients were followed up longitudinally for up to 1 year after kidney transplant. Clinical data collection was completed February 2014. Data analysis was performed from June 1, 2014, to March 5, 2015. Further analysis on baseline and prospective data was performed from June 1, 2017, to July 31, 2019. Main Outcomes and Measures: Cardiovascular functional reserve was objectively quantified using state-of-the-art cardiopulmonary exercise testing in parallel with transthoracic echocardiography. Results: Of the 253 study participants (mean [SD] age, 48.5 [12.7] years; 141 [55.7%] male), 81 were in the KTR group, 85 in the NTWC group, and 87 in the HTC group. At baseline, mean (SD) maximum oxygen consumption (V̇O2max) was significantly lower in the CKD groups (KTR, 20.7 [5.8] mL · min-1 · kg-1; NTWC, 18.9 [4.7] mL · min-1 · kg-1) compared with the HTC group (24.9 [7.1] mL · min-1 · kg-1) (P < .001). Mean (SD) cardiac left ventricular mass index was higher in patients with CKD (KTR group, 104.9 [36.1] g/m2; NTWC group, 113.8 [37.7] g/m2) compared with the HTC group (87.8 [16.9] g/m2), (P < .001). Mean (SD) left ventricular ejection fraction was significantly lower in the patients with CKD (KTR group, 60.1% [8.6%]; NTWC group, 61.4% [8.9%]) compared with the HTC group (66.1% [5.9%]) (P < .001). Kidney transplant was associated with a significant improvement in V̇O2max in the KTR group at 12 months (22.5 [6.3] mL · min-1 · kg-1; P < .001), but the value did not reach the V̇O2max in the HTC group (26.0 [7.1] mL · min-1 · kg-1) at 12 months. V̇O2max decreased in the NTWC group at 12 months compared with baseline (17.7 [4.1] mL · min-1 · kg-1, P < .001). Compared with the KTR group (63.2% [6.8%], P = .02) or the NTWC group (59.3% [7.6%], P = .003) at baseline, transplant was significantly associated with improved left ventricular ejection fraction at 12 months but not with left ventricular mass index. Conclusions and Relevance: The findings suggest that kidney transplant is associated with improved cardiovascular functional reserve after 1 year. In addition, cardiopulmonary exercise testing was sensitive enough to detect a decline in cardiovascular functional reserve in wait-listed patients with CKD. Improved V̇O2max may in part be independent from structural alterations of the heart and depend more on ultrastructural changes after reversal of uremia.

Low serum 25-hydroxyvitamin D is associated with increased bladder cancer risk: A systematic review and evidence of a potential mechanism.

The development of some cancers is associated with vitamin D deficiency. We suggest that reduced conversion of 25-hydroxyvitamin D (25(OH)D) to 1,25-dihydroxyvitamin D (1,25(OH)2D) and the resulting modification of tissue specific immune responses may be key. Non-muscle-invasive bladder cancer is highly immunoresponsive and stimulation of an inflammatory response by intravesical bacillus Calmette-Guerin (BCG) treatment prevents recurrence. To assess the relationship between serum 25(OH)D and bladder cancer risk we conducted a systematic review. To test our hypothesis, the synthesis of 1,25(OH)2D by human bladder epithelial cell lines (T24/83 and RT4) was examined. Studies were identified from Medline, Web of Science, Embase and Cochrane library (limited to English language, humans and 1990-2018). After removal of duplicates, title and abstract review 6 full papers were appraised. Low vitamin D levels were associated with bladder cancer risk in 5/6 of the studies. Both cell lines express the vitamin D receptor, 25-hydroxyvitamin D 1α-hydroxylase (1α-OHase) and 24-hydroxylase (24-OHase) mRNA, which was induced by 1,25(OH)2D. 24-OHase mRNA was also increased by 25(OH)D indicating 1α-OHase activity. Both cell types expressed TLR1,2,4 and the TLR partners MyD88 and CD14mRNA. Cathelicidin mRNA was undetectable in both cell lines but was induced by 1,25(OH)2D and 25(OH)D in RT4 cells. The systematic review demonstrated that bladder cancer risk correlates with serum 25(OH)D levels. In addition, we have shown that transitional epithelial cells express functional vitamin D signaling and can synthesize sufficient 1,25(OH)2D to stimulate a local immune response. We suggest that in order to maintain optimal immune surveillance within the bladder adequate levels of serum 25(OH)D are required for direct synthesis of 1,25(OH)2D by bladder epithelial cells.

Prevalence and treatment of hypovitaminosis D in the haemodialysis population of Coventry.

Low serum 25(OH)D and associated bone and non-bone related problems are not well appreciated in end stage renal disease (ESRD). Vitamin D treatment strategies in the UK currently focus almost exclusively on calcitriol [1,25(OH)2D], alfacalcidol or paricalcitol. In ESRD hypovitaminosis D is associated with bone loss, muscle weakness, falls, fractures and increased inflammation. National guidelines changed in 2014 and now recommend the diagnosis and treatment of low serum 25(OH)D in all patients with glomerular filtration rate (GFR) less than 30ml/min/1.73m2. However as yet there are no standardized guidelines for dosage, frequency and monitoring in ESRD patients. Following a systematic review of the literature we developed a clinical guideline for cholecalciferol supplementation at University Hospitals of Coventry and Warwickshire, UK. The guideline recommends 40,000IU cholecalciferol weekly for patients with 25(OH)D <50nmol/L and 20,000IU weekly for patients with 25(OH)D 50-75nmol/L; to be continued long term unless levels increase to ≥150nmol/L. To date we have measured 25(OH)D levels in 385 in-center haemodialysis patients. Virtually all patients (95%) had serum 25(OH)D levels <75nmol/L (65% deficient, <30nmol/L; 30% insufficient, 30-74nmol/L). Only 5% of patients had optimal levels (≥75nmol/L). Our data indicates that hypovitaminosis D is prevalent in the haemodialysis population in Coventry and Warwickshire and this is likely to reflect UK haemodialysis patients, highlighting the need for a national supplementation guideline.

Arterial Expression of the Calcium-Sensing Receptor Is Maintained by Physiological Pulsation and Protects against Calcification.

UNLABELLED: Vascular calcification (VC) is common in chronic kidney disease (CKD) and contributes to cardiovascular mortality. The calcium-sensing receptor (CaSR) is present in human artery, senses extracellular calcium and may directly modulate VC. OBJECTIVE: to investigate the association between arterial cyclic strain, CaSR expression and VC. METHODS AND RESULTS: human aortic smooth muscle cells (HAoSMC) were cultured under static or strained conditions, with exposure to CaSR agonists, the calcimimetic R568, and after CaSR silencing and over-expression. High extracellular calcium reduced CaSR expression and promoted osteochondrogenic transformation and calcium deposition. This was partially prevented by cyclic strain and exposure to R568. CaSR silencing enhanced calcification and osteochondrogenic transformation, whereas CaSR over-expression attenuated this procalcific response, demonstrating a central role for the CaSR in the response to cyclic strain and regulation of VC. In arterial explants from CKD patients (n = 11) and controls (n = 9), exposure to R568 did not significantly alter calcium deposition, osteochondrogenic markers or total artery calcium content. CONCLUSIONS: physiological mechanical strain is important for arterial homeostasis and may protect arteries from VC. The beneficial effects of cyclic strain may be mediated via the CaSR.

Reduced Cardiovascular Reserve in Chronic Kidney Failure: A Matched Cohort Study.

BACKGROUND: Patients with chronic kidney failure (CKF) experience impaired functional cardiovascular reserve with reduced oxygen consumption at peak exercise (VO(2peak)). No studies have examined whether this is related to impaired cardiovascular compliance as a consequence of loss of adaptive structural alterations, resulting from chronic uremia or hypertension. STUDY DESIGN: Prospective matched-cohort study. SETTING & PARTICIPANTS: We assessed CKF in parallel with patients with essential hypertension but without cardiovascular disease. Patients with CKF were either scheduled for kidney transplantation or transplant waitlisted. 80 patients with CKF and 80 with essential hypertension matched in age, sex, and body mass index were evaluated. 61 patients with CKF (76.3%) were dialysis dependent. PREDICTOR: CKF versus essential hypertension without cardiovascular disease. MEASUREMENTS & OUTCOMES: VO(2peak) was measured during maximal exercise testing. 2-dimensional echocardiography and arterial applanation tonometry were performed prior to exercise testing. To evaluate for the difference in VO(2peak) between study groups, statistically significant predictors of VO(2peak) in multiple regression models were additionally assessed by fitting models comprising the interaction term of patient group with the predictor variable of interest. RESULTS: VO(2peak) was significantly lower in patients with CKF than those with essential hypertension (18.8 vs 24.5 mL/min·kg; P<0.001). Independent predictors of VO(2peak) for CKF included left ventricular (LV) filling pressure (E/mean e'; unstandardized regression coefficient: change in VO(2peak) [in mL/min·kg] per 1-unit change of variable = -5.1) and pulse wave velocity (-4.0); in essential hypertension, these were LV mass index (0.2), LV end-diastolic volume index (0.4), peak heart rate (0.2), and pulse wave velocity (-8.8). The interaction effect of VO(2peak) between patient groups with LV mass index (P<0.001), LV end-diastolic volume index (P<0.001), and peak heart rate (P<0.01) were significantly stronger in the hypertension group, whereby higher values led to greater VO(2peak). LIMITATIONS: Skeletal muscle strength was not assessed. CONCLUSION: This study suggests that maladaptive LV changes, as well as blunted chronotropic response, are important mechanistic factors resulting in reduced cardiovascular reserve in patients with CKF, beyond predominantly vascular changes associated with hypertension.

Characterization of vitamin D production by human ocular barrier cells.

PURPOSE: Vitamin D3 is a secosteroid mainly synthesized from the conversion of the skin precursor 7-dehydrocholesterol (7DHC) to vitamin D3 by ultraviolet (UV) B sunlight. Extrarenal synthesis of vitamin D3 has been reported in many tissues and cells, including barrier sites. This study characterizes the expression of components of vitamin D3 signaling in human ocular barrier cells. METHODS: Primary human scleral fibroblasts (HSF), human corneal endothelial (HCEC-12), nonpigmented ciliary body epithelial (ODM-2), and adult retinal pigment epithelial (ARPE-19) cell lines were analyzed for the expression of vitamin D receptor (VDR), the vitamin D3 activating enzymes 1α-hydroxylase (CYP27B1), 25-hydroxylases (CYP27A1 and CYP2R1), the vitamin D3 inactivating enzyme 24-hydroxylase (CYP24A1), and the endocytic receptors cubilin and megalin using a combination of RT-PCR, immunocytochemistry, and enzyme immunoassay (EIA). RESULTS: The HSF, HCEC-12, ODM-2, and ARPE-19 express mRNA and protein for all vitamin D3 synthesizing and metabolizing components. The cell types tested, except HSF, are able to convert inactive 25-hydroxyvitamin D3 (25[OH]D3) into active 1,25-hydroxyvitamin D3 (1,25[OH]2D3). CONCLUSIONS: This novel study demonstrated that ocular barrier epithelial cells express the machinery for vitamin D3 and can produce 1,25(OH)2D3. We suggest that vitamin D3 might have a role in immune regulation and barrier function in ocular barrier epithelial cells.

Functional cardiovascular reserve predicts survival pre-kidney and post-kidney transplantation.

Exercise intolerance is an important comorbidity in patients with CKD. Anaerobic threshold (AT) determines the upper limits of aerobic exercise and is a measure of cardiovascular reserve. This study investigated the prognostic capacity of AT on survival in patients with advanced CKD and the effect of kidney transplantation on survival in those with reduced cardiovascular reserve. Using cardiopulmonary exercise testing, cardiovascular reserve was evaluated in 240 patients who were waitlisted for kidney transplantation between 2008 and 2010, and patients were followed for ≤5 years. Survival time was the primary endpoint. Cumulative survival for the entire cohort was 72.6% (24 deaths), with cardiovascular events being the most common cause of death (54.2%). According to Kaplan-Meier estimates, patients with AT <40% of predicted peak VO2 had a significantly reduced 5-year cumulative overall survival rate compared with those with AT ≥40% (P<0.001). Regarding the cohort with AT <40%, patients who underwent kidney transplantation (6 deaths) had significantly better survival compared with nontransplanted patients (17 deaths) (hazard ratio, 4.48; 95% confidence interval, 1.78 to 11.38; P=0.002). Survival did not differ significantly among patients with AT ≥40%, with one death in the nontransplanted group and no deaths in the transplanted group. In summary, this is the first prospective study to demonstrate a significant association of AT, as the objective index of cardiovascular reserve, with survival in patients with advanced CKD. High-risk patients with reduced cardiovascular reserve had a better survival rate after receiving a kidney transplant.

Reduced functional measure of cardiovascular reserve predicts admission to critical care unit following kidney transplantation.

BACKGROUND: There is currently no effective preoperative assessment for patients undergoing kidney transplantation that is able to identify those at high perioperative risk requiring admission to critical care unit (CCU). We sought to determine if functional measures of cardiovascular reserve, in particular the anaerobic threshold (VO2AT) could identify these patients. METHODS: Adult patients were assessed within 4 weeks prior to kidney transplantation in a University hospital with a 37-bed CCU, between April 2010 and June 2012. Cardiopulmonary exercise testing (CPET), echocardiography and arterial applanation tonometry were performed. RESULTS: There were 70 participants (age 41.7±14.5 years, 60% male, 91.4% living donor kidney recipients, 23.4% were desensitized). 14 patients (20%) required escalation of care from the ward to CCU following transplantation. Reduced anaerobic threshold (VO2AT) was the most significant predictor, independently (OR = 0.43; 95% CI 0.27-0.68; p<0.001) and in the multivariate logistic regression analysis (adjusted OR = 0.26; 95% CI 0.12-0.59; p = 0.001). The area under the receiver-operating-characteristic curve was 0.93, based on a risk prediction model that incorporated VO2AT, body mass index and desensitization status. Neither echocardiographic nor measures of aortic compliance were significantly associated with CCU admission. CONCLUSIONS: To our knowledge, this is the first prospective observational study to demonstrate the usefulness of CPET as a preoperative risk stratification tool for patients undergoing kidney transplantation. The study suggests that VO2AT has the potential to predict perioperative morbidity in kidney transplant recipients.

Functional expression of TRPV4 channels in human collecting duct cells: implications for secondary hypertension in diabetic nephropathy.

BACKGROUND: The Vanilloid subfamily of transient receptor potential (TRPV) ion channels has been widely implicated in detecting osmotic and mechanical stress. In the current study, we examine the functional expression of TRPV4 channels in cell volume regulation in cells of the human collecting duct. METHODS: Western blot analysis, siRNA knockdown, and microfluorimetry were used to assess the expression and function of TRPV4 in mediating Ca²âº-dependent mechanical stimulation within a novel system of the human collecting duct (HCD). RESULTS: Native and siRNA knockdown of TRPV4 protein expression was confirmed by western blot analysis. Touch was used as a cell-directed surrogate for osmotic stress. Mechanical stimulation of HCD cells evoked a transient increase in [Ca²âº](i) that was dependent upon thapsigargin-sensitive store release and Ca²âº influx. At 48 hrs, high glucose and mannitol (25 mM) reduced TRPV4 expression by 54% and 24%, respectively. Similar treatment doubled SGK1 expression. Touch-evoked changes were negated following TRPV4 knockdown. CONCLUSION: Our data confirm expression of Ca²âº-dependent TRPV4 channels in HCD cells and suggest that a loss of expression in response to high glucose attenuates the ability of the collecting duct to exhibit regulatory volume decreases, an effect that may contribute to the pathology of fluid and electrolyte imbalance as observed in diabetic nephropathy.

Determining resource intensity weights in ambulatory chemotherapy related to nursing workload.

Ontario cancer programs aim to deliver high-quality nursing care and treatment that is safe for patients and staff. The reality of health care is that financial constraints, inherent in the delivery of care, require that funding mechanisms count not only the cost of drugs, but factors such as pharmacy and nursing human resource costs. While some organizations have developed patient classification systems to measure nursing intensity and workload, these systems apply primarily to inpatient populations, and are fraught with numerous challenges, such as the need for nurses to document to justify the workload required for care. The purpose of this paper is to outline the methodology and engagement of nurses to develop regimen-based resource intensity weights that can be applied to ambulatory chemotherapy suites. The methodology included determination of workload related to nursing time to prepare, teach, counsel and assess patients, as well as time to gather supplies, access lines, monitor, manage adverse reactions, manage symptoms and document care. Resource intensity weights provide better measures of the complexity of care required by cancer patients in ambulatory settings.

TGF-beta1 mediates glucose-evoked up-regulation of connexin-43 cell-to-cell communication in HCD-cells.

BACKGROUND/AIMS: In the current study we examined if the multifunctional cytokine TGF-beta1 mediated glucose-evoked increases in connexin-43(Cx43)-mediated intercellular communication in cells of the human collecting duct (HCD). METHODS: RT-PCR and western blot analysis were used to confirm mRNA and protein expression of TGF-beta1 and Cx43 in HCD-cells. The effect of TGF-beta1 and high glucose (25 mM) on Cx43 protein expression, cytoskeletal organisation and cell-cell communication was determined in the presence/absence of TGF-beta1 specific immuno-neutralising antibodies. Functional cell-cell communication was determined using Ca2+-microfluorimetry. RESULTS: At 24 hrs, high glucose (25 mM) significantly increased Cx43 mRNA and protein expression. Changes were mimicked by TGF-beta1 (2 ng/ml) at low glucose (5 mM). Both high glucose and TGF-beta1 mediated changes were completely reversed by a pan-specific immuno-neutralising antibody to TGF-beta. Furthermore, high glucose-evoked changes were inhibited by a TGF-beta1-specific monoclonal antibody. Mannitol (25 mM), an osmotic control for high glucose, failed to alter Cx43 expression. TGF-beta1 evoked changes in Cx43 expression were biphasic. An early (4-8 hr) transient decrease in expression was followed by an increase in protein expression (12-24 hr). The decrease in Cx43 expression was paralleled by a transient reorganisation of the actin cytoskeleton, whilst increased Cx43 expression at 24 hrs coincided with a TGF-beta1 specific increase in touch-evoked transmission of Ca2+-signals between coupled cells. CONCLUSIONS: High glucose evoked a TGF-beta1 mediated increase in Cx43 expression and gap-junction mediated cell-cell communication in HCD-cells. These changes may maintain epithelial integrity of the collecting duct following hyperglycaemic assault as observed in diabetes.

Expression and role of the calcium-sensing receptor in the blood vessel wall.

The calcium-sensing receptor (CaSR), which is involved in systemic calcium homeostasis, has also been found to be functionally expressed on cells of the vascular wall. Its activation on perivascular nerves and endothelial cells has been shown to regulate arterial tone, peripheral vascular resistance and possibly local tissue perfusion. The expression of the CaSR on immune cells involved in vascular inflammation, such as macrophages, and its increased expression in inflammation indicates the central role extracellular calcium plays in vascular inflammation and repair. Further detailed analysis will clarify the role the vascular CaSR plays as a therapeutic target for complex disease conditions such as hypertension, tissue hypoperfusion, atherosclerosis and vascular calcification.

Extracellular calcium-sensing receptor mediated signalling is involved in human vascular smooth muscle cell proliferation and apoptosis.

Calcium-sensing receptor (CaSR) plays key role in vascular calcification in patients with chronic kidney disease (CKD). We investigated the role of CaSR in regulating smooth muscle cell (SMC) proliferation and apoptosis. Incubation with 300 microM neomycin (CaSR agonist) resulted in 7.5-fold (p<0.05) increase in ERK1,2 phosphorylation. It was reduced (p<0.01) by 10 microM PD98059 (MEK1 inhibitor), indicating that CaSR agonist-induced effects were mediated via MEK1/ERK1,2 pathway. ERK1,2 phosphorylation was abolished by 5 microM U73122 (PLC inhibitor), indicating that PLC signalling was crucial for MEK1/ERK1,2 activation. Confirming PLC activation, inositol triphosphate (IP3) production was increased by neomycin/gentamycin (p<0.05) and reduced by U73122. To confirm that ERK1,2 and PLC signalling were mediated via CaSR, Human Aortic SMC (HAoSMC) were transfected with CaSR siRNA. CaSR knockdown resulted in lower ERK1,2 neomycin response and IP3 production (p<0.01). Neomycin increased HAoSMC proliferation >3-fold, which was reduced in CaSR knockdown cells (p<0.01) and further inhibited by PD98059 and U73122 (p<0.05). Apoptosis was not affected by neomycin treatment. U73122 produced 3.5-fold increase in HAoSMC apoptosis, which was further increased by CaSR knockdown (5-fold, p<0.05). In conclusion, stimulation of CaSR leads to activation of MEK1/ERK1,2 and PLC pathways and up-regulation of cell proliferation. CaSR-mediated PLC activation is important for SMC survival and protection against apoptosis.

Reduction of the vitamin D hormonal system in kidney disease is associated with increased renal inflammation.

To examine any potential role for 1,25-dihydroxyvitamin D (1,25(OH)2D) in inflammation associated with chronic kidney disease we measured vitamin D metabolites, markers of inflammation and gene expression in 174 patients with a variety of kidney diseases. Urinary MCP-1 protein and renal macrophage infiltration were each significantly but inversely correlated with serum 1,25(OH)2D levels. Logistic regression analysis with urinary MCP-1 as binary outcome showed that a 10-unit increase in serum 1,25(OH)2D or 25OHD resulted in lower renal inflammation. Analysis of 111 renal biopsies found that renal injury was not associated with a compensatory increase in mRNA for the vitamin D-activating enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), its catabolic counterpart 24-hydroxylase, or the vitamin D receptor. There was, however, a significant association between tissue MCP-1 and CYP27B1. Patients with acute renal inflammation had a significant increase in urinary and tissue MCP-1, macrophage infiltration, and macrophage and renal epithelial CYP27B1 expression but significantly lower levels of serum 1,25(OH)2D in comparison to patients with chronic ischemic disease despite similar levels of renal damage. In vitro, 1,25(OH)2D attenuated TNFalpha-induced MCP-1 expression by human proximal tubule cells. Our study indicates that renal inflammation is associated with decreased serum vitamin D metabolites and involves activation of the paracrine/autocrine vitamin D system.

Serum and glucocorticoid regulated kinase and disturbed renal sodium transport in diabetes.

Diabetes is associated with a number of side effects including retinopathy, neuropathy, nephropathy and hypertension. Recent evidence has shown that serum and glucocorticoid regulated kinase-1 (SGK1) is increased in models of diabetic nephropathy. While clearly identified as glucocorticoid responsive, SGK1 has also been shown to be acutely regulated by a variety of other factors. These include insulin, hypertonicity, glucose, increased intracellular calcium and transforming growth factor-beta, all of which have been shown to be increased in type II diabetes. The principal role of SGK1 is to mediate sodium reabsorption via its actions on the epithelial sodium channel (now known as sodium channel, nonvoltage-gated 1). Small alterations in the sodium resorptive capacity of the renal epithelia may have dramatic consequences for fluid volume regulation, and SGK1 maybe responsible for the development of hypertension associated with diabetes. This short commentary considers the evidence that supports the involvement of SGK1 in diabetic hypertension, but also discusses how aberrant sodium reabsorption may account for the cellular changes seen in the nephron.

Phorbol ester-stimulated NF-kappaB-dependent transcription: roles for isoforms of novel protein kinase C.

Since protein kinase C (PKC) isoforms are variously implicated in the activation of NF-kappaB, we have investigated the role of PKC in the activation of NF-kappaB-dependent transcription by the diacyl glycerol (DAG) mimetic, phorbol 12-myristate 13-acetate (PMA), and by tumour necrosis factor (TNF) alpha in pulmonary A549 cells. The PKC selective inhibitors, Ro31-8220, Gö6976, GF109203X and Gö6983, revealed no effect on TNFalpha-induced NF-kappaB DNA binding and a similar lack of effect on serine 32/36 phosphorylated IkappaBalpha and the loss of total IkappaBalpha indicates that activation of the core IKK-IkappaBalpha-NF-kappaB cascade by TNFalpha does not involve PKC. In contrast, differential sensitivity of an NF-kappaB-dependent reporter to Ro31-8220, Gö6976, GF109203X and Gö6983 (EC(50)s 0.46 microM, 0.34 microM, >10 microM and >10 microM respectively) suggests a role for protein kinase D in transcriptional activation by TNFalpha. Compared with TNFalpha, PMA weakly induces NF-kappaB DNA binding and this effect was not associated with serine 32/36 phosphorylation of IkappaBalpha. However, PMA-stimulated NF-kappaB DNA binding was inhibited by Ro31-8220 (10 microM), GF109203X (10 microM) and Gö6983 (10 microM), but not by Gö6976 (10 microM), suggesting a role for novel PKC isoforms. Furthermore, a lack of positive effect of calcium mobilising agents on both NF-kappaB DNA binding and on transcriptional activation argues against major roles for classical PKCs. This, combined with the ability of both GF109203X and Gö6983 to prevent enhancement of TNFalpha-induced NF-kappaB-dependent transcription by PMA, further indicates a role for novel PKCs in NF-kappaB transactivation. Finally, siRNA-mediated knockdown of PKCdelta and epsilon expression did not affect TNFalpha-induced NF-kappaB-dependent transcription. However, knockdown of PKCdelta expression significantly inhibited PMA-stimulated luciferase activity, whereas knockdown of PKCepsilon was without effect. Furthermore, combined knockdown of PKCdelta and epsilon revealed an increased inhibitory effect on PMA-stimulated NF-kappaB-dependent transcription suggesting that PMA-induced NF-kappaB-dependent transcription is driven by novel PKC isoforms, particularly PKCdelta and epsilon.

Extracellular calcium-sensing receptor is functionally expressed in human artery.

Accelerated medial calcification is a major cause of premature cardiovascular mortality in patients with chronic kidney disease (CKD). Evidence suggests that extracellular concentration of Ca2+ and vascular smooth muscle cells may play a pivotal role in the pathogenesis of vascular calcification. The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that is expressed in a range of tissues, but characterization of its expression and function in the cardiovascular system is limited. Here we report the expression of CaSR mRNA (RT-PCR) and protein (Western blotting and immunocytochemistry) in human aortic smooth muscle cells (HAoSMC). Treatment of HAoSMC with Ca2+ (0-5 mM; 0-30 min) or the CaSR agonists gentamycin and neomycin (0-300 microM; 0-30 min) resulted in a dose- and time-dependent phosphorylation of ERK1/2. Gentamycin- and neomycin-mediated ERK1/2 stimulation was inhibited by pretreatment with PD-98059, an ERK-activating kinase 1 (MEK1) inhibitor, confirming specificity of the observed effects. ERK1/2 activation was inhibited in HAoSMC, with CaSR expression knocked down by transfection with specific small-interference RNA, which confirmed that the observed neomycin/gentamycin-induced MEK1/ERK1/2 activation was mediated via the CaSR. CaSR mRNA and protein were also expressed in large and small arteries from normal subjects (kidney donors) and patients with end-stage renal disease (ESRD). The CaSR was detected in smooth muscle and endothelial cells. Expression was significantly lower in arteries from ESRD patients. In conclusion, these data not only demonstrate the presence of a functional CaSR in human artery but show a correlation between CaSR expression and progression of CKD.

High glucose up-regulates ENaC and SGK1 expression in HCD-cells.

BACKGROUND/AIM: Diabetic nephropathy is associated with progressive renal damage, leading to impaired function and end-stage renal failure. Secondary hypertension stems from a deranged ability of cells within the kidney to resolve and appropriately regulate sodium resorption in response to hyperglycaemia. However, the mechanisms by which glucose alters sodium re-uptake have not been fully characterised. METHODS: Here we present RT-PCR, western blot and immunocytochemistry data confirming mRNA and protein expression of the serum and glucocorticoid inducible kinase (SGK1) and the alpha conducting subunit of the epithelial sodium channel (ENaC) in a model in vitro system of the human cortical collecting duct (HCD). We examined changes in expression of these elements in response to glucose challenge, designed to mimic hyperglycaemia associated with type 2 diabetes mellitus. Changes in Na+ concentration were assessed using single-cell microfluorimetry. RESULTS: Incubation with glucose, the Ca2+-ionophore ionomycin and the cytokine TGF-beta1 were all found to evoke significant and time-dependent increases in both SGK1 and alphaENaC protein expression. These molecular changes were correlated to an increase in Na+-uptake at the single-cell level. CONCLUSION: Together these data offer a potential explanation for glucose-evoked Na+-resorption and a potential contributory role of SGK1 and ENaCs in development of secondary hypertension, commonly linked to diabetic nephropathy.