Rosemary Bland

Differential regulation of vitamin D receptor and its ligand in human monocyte-derived dendritic cells

The functions of dendritic cells (DCs) are tightly regulated such that protective immune responses are elicited and unwanted immune responses are prevented. 1α25-dihydroxyvitamin D3 (1α25(OH)2D3) has been identified as a major factor that inhibits the differentiation and maturation of DCs, an effect dependent upon its binding to the nuclear vitamin D receptor (VDR). Physiological control of 1α25(OH)2D3 levels is critically dependent upon 25-hydroxyvitamin D3-1α-hydroxylase (1αOHase), a mitochondrial cytochrome P450 enzyme that catalyzes the conversion of inactive precursor 25-hydroxyvitamin D3 (25(OH)D3) to the active metabolite 1α25(OH)2D3. Using a human monocyte-derived DC (moDC) model, we have examined the relationship between DC VDR expression and the impact of exposure to its ligand, 1α25(OH)2D3. We show for the first time that moDCs are able to synthesize 1α25(OH)2D3 in vitro as a consequence of increased 1αOHase expression. Following terminal differentiation induced by a diverse set of maturation stimuli, there is marked transcriptional up-regulation of 1αOHase leading to increased 1αOHase enzyme activity. Consistent with this finding is the observation that the development and function of moDCs is inhibited at physiological concentrations of the inactive metabolite 25(OH)D3. In contrast to 1αOHase, VDR expression is down-regulated as monocytes differentiate into immature DCs. Addition of 1α25(OH)2D3 to moDC cultures at different time points indicates that its inhibitory effects are greater in monocyte precursors than in immature DCs. In conclusion, differential regulation of endogenous 1α25(OH)2D3 ligand and its nuclear receptor appear to be important regulators of DC biology and represent potential targets for the manipulation of DC function.

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.

Phorbol ester-stimulated NF-κB-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 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 300μM neomycin (CaSR agonist) resulted in 7.5-fold (p<0.05) increase in ERK1,2 phosphorylation. It was reduced (p<0.01) by 10μM PD98059 (MEK1 inhibitor), indicating that CaSR agonist-induced effects were mediated via MEK1/ERK1,2 pathway. ERK1,2 phosphorylation was abolished by 5μM 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.

Expression of 25-hydroxyvitamin D3-1alpha-hydroxylase along the nephron: new insights into renal vitamin D metabolism.

Renal synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a pivotal step in calcium and phosphate homeostasis. Production of 1,25(OH)2D3 is catalyzed by the mitchondrial cytochrome P450, 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-HYD). As a consequence of the tight regulation of vitamin D metabolism during normal physiology, studies of the expression and regulation of 1alpha-HYD have proved remarkably difficult. However, the recent cloning of the gene for 1alpha-HYD has enabled a more comprehensive analysis of the tissue distribution of 1alpha-HYD, as well as the mechanisms involved in controlling 1,25(OH)2D3 production. In particular, an understanding of site-specific expression and regulation of 1alpha-HYD along the nephron might help to elucidate a more versatile role for 1,25(OH)2D3 in renal physiology.

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 α 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-β1 were all found to evoke significant and time-dependent increases in both SGK1 and αENaC 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.

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.

TGF-β1 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-β1 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-β1 and Cx43 in HCD-cells. The effect of TGF-β1 and high glucose (25mM) on Cx43 protein expression, cytoskeletal organisation and cell-cell communication was determined in the presence/absence of TGF-β1 specific immuno-neutralising antibodies. Functional cell-cell communication was determined using Ca2+-microfluorimetry. Results: At 24hrs, high glucose (25mM) significantly increased Cx43 mRNA and protein expression. Changes were mimicked by TGF-β1 (2ng/ml) at low glucose (5mM). Both high glucose and TGF-β1 mediated changes were completely reversed by a pan-specific immuno-neutralising antibody to TGF-β. Furthermore, high glucose-evoked changes were inhibited by a TGF-β1-specific monoclonal antibody. Mannitol (25mM), an osmotic control for high glucose, failed to alter Cx43 expression. TGF-β1 evoked changes in Cx43 expression were biphasic. An early (4-8hr) transient decrease in expression was followed by an increase in protein expression (12-24hr). The decrease in Cx43 expression was paralleled by a transient reorganisation of the actin cytoskeleton, whilst increased Cx43 expression at 24hrs coincided with a TGF-β1 specific increase in touch-evoked transmission of Ca2+-signals between coupled cells. Conclusions: High glucose evoked a TGF-β1 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.

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.