Assessment of differentially methylated loci in individuals with end-stage kidney disease attributed to diabetic kidney disease: an exploratory study.

BACKGROUND: A subset of individuals with type 1 diabetes mellitus (T1DM) are predisposed to developing diabetic kidney disease (DKD), the most common cause globally of end-stage kidney disease (ESKD). Emerging evidence suggests epigenetic changes in DNA methylation may have a causal role in both T1DM and DKD. The aim of this exploratory investigation was to assess differences in blood-derived DNA methylation patterns between individuals with T1DM-ESKD and individuals with long-duration T1DM but no evidence of kidney disease upon repeated testing to identify potential blood-based biomarkers. Blood-derived DNA from individuals (107 cases, 253 controls and 14 experimental controls) were bisulphite treated before DNA methylation patterns from both groups were generated and analysed using Illumina's Infinium MethylationEPIC BeadChip arrays (n = 862,927 sites). Differentially methylated CpG sites (dmCpGs) were identified (false discovery rate adjusted p ≤ × 10-8 and fold change ± 2) by comparing methylation levels between ESKD cases and T1DM controls at single site resolution. Gene annotation and functionality was investigated to enrich and rank methylated regions associated with ESKD in T1DM. RESULTS: Top-ranked genes within which several dmCpGs were located and supported by functional data with methylation look-ups in other cohorts include: AFF3, ARID5B, CUX1, ELMO1, FKBP5, HDAC4, ITGAL, LY9, PIM1, RUNX3, SEPTIN9 and UPF3A. Top-ranked enrichment pathways included pathways in cancer, TGF-ß signalling and Th17 cell differentiation. CONCLUSIONS: Epigenetic alterations provide a dynamic link between an individual's genetic background and their environmental exposures. This robust evaluation of DNA methylation in carefully phenotyped individuals has identified biomarkers associated with ESKD, revealing several genes and implicated key pathways associated with ESKD in individuals with T1DM.

Lipoxins: update and impact of endogenous pro-resolution lipid mediators.

Lipoxins (LXs) are endogenously produced eicosanoids that are typically generated by transcellular biosynthesis. These trihydroxytetraene-containing lipid mediators and their stable synthetic analogues possess a wide spectrum of anti-inflammatory and pro-resolution bioactions both in vitro and in vivo. More recently, LXs have emerged as potential anti-fibrotic mediators that may influence pro-fibrotic cytokines and matrix-associated gene expression in response to platelet-derived growth factor (PDGF). Here we review the biosynthesis, metabolism and bioactions of LXs and LX analogues and their therapeutic potential.

Melatonin receptors are for the birds: molecular analysis of two receptor subtypes differentially expressed in chick brain.

Two receptors (CKA and CKB) of the G protein-coupled melatonin receptor family were cloned from chick brain. CKA encodes a protein that is 80% identical at the amino acid level to the human Mel1a melatonin receptor and is thus designated the chick Mel1a melatonin receptor. CKB encodes a protein that is 80% identical to the Xenopus melatonin receptor and defines a new receptor subtype, the Mel1c melatonin receptor, which is distinct from the Mel1a and Mel1b melatonin receptor subtypes. A melatonin receptor family consisting of three subtypes is supported by PCR cloning of distinct melatonin receptor fragments from Xenopus and zebrafish. Expression of CKA and CKB results in similar ligand binding and functional characteristics. The widespread distribution of CKA and CKB mRNA in brain provides a molecular substrate for the profound actions of melatonin in birds.

Validating the association between plasma tumour necrosis factor receptor 1 levels and the presence of renal injury and functional decline in patients with Type 2 diabetes.

AIMS: Elevated plasma soluble tumour necrosis factor receptor 1 (TNFR1) predicts long-term progression of chronic kidney disease. We investigated the association between elevated TNFR1 and the presence of renal disease in patients with Type 2 diabetes mellitus registering a haemoglobin A1c (HbA1c) >48mmol/mol despite medical therapy. METHODS: Using sensitivity, specificity and regression analyses we interrogated the association between plasma TNFR1 and presence of chronic kidney disease as assessed by the presence of microalbuminuria and/or an estimated glomerular filtration rate of less than 60ml/min/1.73m2 (stages 3-5 chronic kidney disease). The association of TNFR1 with C-reactive protein and leptin-adiponectin ratio as plasma markers of systemic inflammation and adipose stress respectively was also investigated. RESULTS: Upper quartile TNFR1 is independently associated with elevated urinary albumin-creatinine ratios, reductions in eGFR and strongly predicts the presence of stages 3-5 chronic kidney disease in regression modelling. Elevated TNFR1 levels are associated with increased plasma C-reactive protein and augmented leptin-adiponectin ratio. CONCLUSIONS: Our study confirms plasma TNFR1 as a surrogate of renal structural and functional impairment in patients with type 2 diabetes mellitus. Association of TNFR1 with markers of systemic inflammation and adipose stress indicates that TNFR1 may be a biomarker of these processes as components of the pathogenesis of diabetic kidney disease.

Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B-mediated phosphorylation and cytoplasmic translocation of p27(Kip-1).

Connective tissue growth factor (CTGF/CCN2) is a 38-kDa secreted protein, a prototypic member of the CCN family, which is up-regulated in many diseases, including atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that CTGF can cause actin disassembly with concurrent down-regulation of the small GTPase Rho A and proposed an integrated signaling network connecting focal adhesion dissolution and actin disassembly with cell polarization and migration. Here, we further delineate the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The functional response of mesangial cells to treatment with CTGF was associated with the phosphorylation of Akt/protein kinase B (PKB) and resultant phosphorylation of a number of Akt/PKB substrates. Two of these substrates were identified as FKHR and p27(Kip-1). CTGF stimulated the phosphorylation and cytoplasmic translocation of p27(Kip-1) on serine 10. Addition of the PI-3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleukin (IL)-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate prevented p27(Kip-1) phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27(Kip-1) colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27(Kip-1), revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27(Kip-1) cytoplasmic localization; mutation of the threonine 157 site prevented cytoplasmic localization, protected against actin disassembly and inhibited cell migration. CTGF also stimulated an increased association between Rho A and p27(Kip-1). Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27(Kip-1) activation results in uncoupling of the Rho A/LIM kinase/cofilin pathway. Confirming the central role of Akt/PKB, CTGF-stimulated actin depolymerization only in wild-type mouse embryonic fibroblasts (MEFs) compared to Akt-1/3 (PKB alpha/gamma) knockout MEFs. These data reveal important mechanistic insights into how CTGF may contribute to mesangial cell dysfunction in the diabetic milieu and sheds new light on the proposed role of p27(Kip-1) as a mediator of actin rearrangement.

Isoform-specific redistribution of protein kinase C in living cells.

We have used confocal laser scanning microscopy to determine the dynamics of distribution of activated protein kinase C (PKC) in living Madin Darby canine kidney (MDCK) cells. Using fluorescently tagged phorbol myristate acetate (PMA) as a probe for PKC we have demonstrated its distribution in association with the cell periphery and with the nucleus. Dual labeling experiments using PKC alpha and PKC beta II specific antisera indicate that activated PKC alpha is found in association with the periphery whereas activated PKC beta II is translocated to the nucleus. We have demonstrated increased activity of PKC in nuclear fractions isolated from cells treated with PMA and other PKC activators. These data indicate that upon activation individual isoforms of PKC translocate to different subcellular locations where they are likely to mediate different actions.

Lipoxins: revelations on resolution.

Lipoxins (LXs) are endogenously produced eicosanoids typically generated during cell-cell interactions. In this article, the compelling evidence from in vitro and in vivo model systems that LXs actively promote the resolution of inflammation is reviewed. Of particular interest are indications that stable synthetic analogues of LXs and aspirin-triggered 15-epi-LXs can mimic many of the desirable anti-inflammatory, "pro-resolution" actions of native LXs. Given the enhanced stability and efficacy of these compounds a role as novel anti-inflammatory therapeutics is proposed.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization.

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

Gremlin gene expression in bovine retinal pericytes exposed to elevated glucose.

AIM: To assess the influence of high extracellular glucose on the expression of the bone morphogenetic protein (BMP) antagonist, gremlin, in cultured bovine retinal pericytes (BRPC). METHODS: BRPC were cultured under conditions of 5 mM and 30 mM d-glucose for 7 days and total RNA was isolated. Gremlin mRNA levels were correlated, by RT-PCR, with other genes implicated in the pathogenesis of diabetic retinopathy and the signalling pathways in high glucose induced gremlin expression were probed using physiological inhibitors. Gremlin expression was also examined in the retina of streptozotocin induced diabetic mice. RESULTS: High glucose stimulated a striking increase in BRPC gremlin mRNA levels in parallel with increases in mRNA for the growth factors vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFbeta), and connective tissue growth factor (CTGF) and changes in other genes including fibronectin and plasminogen activator inhibitor-1 (PAI-1). High glucose triggered gremlin expression was modulated by anti-TGFbeta antibody, by the uncoupler of oxidative phosphorylation, CCCP, and by inhibition of MAP-kinase (MAPK) activation. Striking gremlin expression was observed in the outer retina of diabetic mice and also at the level of the vascular wall. CONCLUSIONS: Gremlin gene expression is induced in BRPC in response to elevated glucose and in the retina of the streptozotocin induced diabetic mouse. Its expression is modulated by hyperglycaemic induction of the MAPK, reactive oxygen species, and TGFbeta pathways, all of which are reported to have a role in diabetic fibrotic disease. This implicates a role for gremlin in the pathogenesis of diabetic retinopathy.

The Mel1a melatonin receptor is coupled to parallel signal transduction pathways.

The recent cloning of a family of high affinity melatonin receptors has provided us with a unique opportunity to define the signal transduction pathways used by these receptors. We have studied signaling through the human Mel1a receptor subtype by stable expression of receptor complementary DNA in NIH 3T3 cells. Our data indicate that the human Mel1a receptor is coupled to inhibition of forskolin-stimulated cAMP accumulation by a pertussis toxin-sensitive G protein. Although melatonin alone is without effect on phosphoinositide hydrolysis, it potentiates the effects of PGF2 alpha stimulation on phospholipase C activation. Melatonin potentiates arachidonate release stimulated by PGF2 alpha and by ionomycin. The effects of melatonin on arachidonate release are sensitive to inhibition of protein kinase C. They are independent of the effects of melatonin on cAMP and do not appear to involve activation of mitogen-activated protein kinase. The effects of melatonin on both phosphoinositide hydrolysis and arachidonate release are sensitive to pertussis toxin treatment. Thus, we show that the melatonin signal is transduced by parallel pathways involving inhibition of adenylyl cyclase and potentiation of phospholipase activation.

Structure, characterization, and expression of the gene encoding the mouse Mel1a melatonin receptor.

Recently, a distinct family of G protein-coupled receptors has been cloned that mediates the biological effects of melatonin. Of two sub-types cloned from mammals (Mel1a and Mel1b), the Mel1a receptor appears to mediate the circadian and reproductive effects of the hormone. We now report the cloning, characterization, and expression of the gene encoding the Mel1a receptor in mice. The receptor gene is composed of two exons, separated by an intron of greater than 13 kilobases. Exon 1 encodes the entire 5'-untranslated region and the coding region through the first cytoplasmic loop. Exon 2 encodes the rest of the coding region and the entire 3'-untranslated region. 5'-Rapid amplification of complementary DNA ends and ribonuclease protection analyses show that the major transcription start site is 103 nucleotides upstream of the translation start codon. Sequence analysis of 1.1 kilobases of the 5'-flanking region reveals that it does not contain TATA or CAAT boxes. The 5'-flanking region drives luciferase expression 114-fold over basal levels in a murine retinal cell line that endogenously expresses the Mel1a receptor. The mouse receptor binds 2-[125]iodomelatonin with high affinity (K(d) = 55.6 pM) when expressed transiently in COS-7 cells. In situ hybridization studies establish that Mel1a receptor messenger RNA is expressed in the hypothalamic suprachiasmatic nuclei and hypophyseal pars tuberalis, presumed sites of the circadian and some of reproductive actions of melatonin, respectively. These results provide information on Mel1a receptor gene structure essential for designing transgenic and gene knock-out studies and analyzing the transcriptional regulation of receptor gene expression.

Induction of c-fos in pituitary cells by thyrotrophin-releasing hormone and phorbol 12-myristate 13-acetate depends upon Ca2+ influx.

The role of cytosolic free Ca2+ ([Ca2+]i) in the induction of the immediate early gene c-fos by TRH or by phorbol 12-myristate 13-acetate (PMA) was studied in the clonal pituitary cell line GH4C1. It was found that c-fos mRNA levels were rapidly and transiently increased by TRH at physiological concentrations (1-100 nM). The effect of TRH was dependent on a rise in [Ca2+]i, and TRH stimulation of Ca2+ influx was essential for c-fos induction. Cell depolarization with K+, which produces a [Ca2+]i rise by soliciting Ca2+ influx via voltage-gated Ca2+ channels, was insufficient to induce c-fos. Blockade or downregulation of protein kinase C (PKC) strongly attenuated TRH stimulation of c-fos expression. Direct stimulation of PKC by PMA raised c-fos mRNA levels, but only under conditions permitting Ca2+ influx. We conclude that TRH induces c-fos mRNA by a mechanism dependent on PKC activation and on Ca2+ influx. The essential role of Ca2+ influx for PMA stimulation of c-fos mRNA suggests a novel pathway linking PKC stimulation to early gene expression.

Lipoxins: novel anti-inflammatory therapeutics?

Lipoxins (LX) are endogenously produced eicosanoids that promote the resolution of inflammation. Here we review the accumulating evidence that LX act as potent modulators of leukocyte trafficking in model systems in vitro and in vivo. Of particular interest are indications that aspirin-triggered epi-LX and synthetic LX combine many of the desirable anti-inflammatory actions of LX with enhanced stability and bioavailability. Such synthetic analogs have potential as novel therapeutics. Furthermore, the bioactivities of the aspirin triggered LX may account for some of the well-documented prostaglandin-independent anti-inflammatory actions of aspirin.

Acute hypomagnesaemia complicating the treatment of meconium ileus equivalent in cystic fibrosis.

Meconium ileus equivalent (MIE) is a common and often recurrent complication in adolescent and adult patients with cystic fibrosis (CF). MIE is characterized by partial or complete bowel obstruction, resulting from abnormally viscid mucofaeculant material in the terminal ileum and right colon. Patients present with recurrent abdominal pain, intestinal obstruction, and/or a palpable faecal mass. Conventional treatment consists of the oral and rectal administration of the mucolytic agent N-acetylcysteine, and hypertonic solutions of sodium diatrizoate. We describe the occurrence of acute decreases in plasma magnesium in all of seven patients treated with this regimen with marked hypomagnesaemia (less than 0.70 mmol/l) in four of the seven patients. No changes in plasma sodium, potassium, or calcium were observed.

Investigation of aminoglycoside nephrotoxicity in cystic fibrosis patients.

Cystic fibrosis patients are a special risk group for nephrotoxic effects of amino-glycosides (AG). The traditional methods of assessing renal damage are very insensitive, and toxicity is not detected until serious functional damage is evident. The aims of the present study were to monitor early markers of nephrotoxicity in cystic fibrosis patients treated with gentamicin. Urinary excretion of beta-N-acetylglucosaminase (beta-NAG, a lysosomal enzyme) and low molecular weight proteins as identified on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were prospectively studied in cystic fibrosis outpatients and inpatients. Urinary excretions of beta-NAG and low molecular weight proteins were normal in cystic fibrosis patients not being treated with gentamicin. All patients being treated with intravenous gentamicin (average dose, 2.5 g over 9 days) showed markedly elevated urinary excretion of beta-NAG. SDS-PAGE of serial urine samples from CF patients during intravenous AG therapy showed increasing excretion of low molecular weight proteins. Protein excretion profiles from control subjects showed no change over equivalent time periods. CF patients receiving nebulized AG did not show altered protein excretion. beta-NAG excretion patterns were similar to changes detected in the SDS-PAGE system. No gross nephrotoxicity developed in these patients. Blood urea nitrogen, plasma creatinine, and plasma magnesium concentrations remained within normal limits. The pattern of protein excretion on SDS-PAGE was consistent with renal tubular rather than glomerular damage. The SDS-PAGE system provides an additional means of monitoring early subclinical nephrotoxicity of AG in CF patients.

Protein kinases C: potential targets for intervention in diabetic nephropathy.

Protein kinases C are a family of serine threonine protein kinases that play key roles in extracellular signal transduction. Inappropriate activation of protein kinase C has been implicated in the pathophysiology of many diseases, including diabetes mellitus. Indeed, protein kinase C activation may contribute not only to the pathogenesis of diabetic complications such as nephropathy and retinopathy, but also to insulin resistance. Growing awareness that protein kinase C isoforms subserve specific subcellular functions has led to the development of isoform-specific inhibitors, which may be useful investigational tools and therapeutic agents for attenuating the effects of inappropriate protein kinase C activity. Here we review the role played by protein kinases C in diabetic nephropathy and the recent progress that has been made to modulate its activity using specific inhibitors.

ras-transformation of MDCK cells alters responses to phorbol ester without altering responses to bradykinin.

The results of studies to evaluate the hypothesis that the 21 kDa GTP-binding protein derived from the ras oncogene is involved in regulation and coupling of hormone receptors to phospholipase activity have thus far been inconsistent. We therefore examined the effect of H-ras transformation on basal, tumor-promoting phorbol ester (TPA)-stimulated, and bradykinin-mediated phospholipid hydrolysis in Madin Darby canine kidney cells (MDCK) by comparing H-ras-transformed MDCK cells (MDCK-RAS) to two non-transformed strains of MDCK cells (MDCK-D1 and MDCK-ATCC). In unstimulated MDCK-RAS, diacylglycerol (DAG), inositol phosphate accumulation, and choline phosphate release were increased while arachidonic acid and arachidonic acid metabolite (AA) release was not increased, suggesting that ras transformation increased phospholipase C activity. Protein kinase C (PK-C) activity was decreased, and specific binding of [3H]phorbol ester was reduced in MDCK-RAS relative to the non-transformed MDCK cells suggesting that elevated DAG may activate and thereby down-regulate PK-C. Consistent with this finding in MDCK-RAS, TPA-stimulated AA release and subsequent prostaglandin E2 production were decreased, while TPA-stimulated choline phosphate release was increased. Bradykinin receptor-stimulated phospholipid hydrolysis in MDCK-RAS was similar to that of non-transformed cells, suggesting that the ras-derived protein does not directly couple bradykinin receptors to phospholipases in MDCK cells. However, the ability of TPA-treatment to inhibit bradykinin-stimulated phosphoinositide hydrolysis and enhance bradykinin-stimulated AA release was attenuated in MDCK-RAS. Additionally, in MDCK-RAS the conversion of arachidonic acid to prostaglandin E2 was substantially reduced. We conclude that ras transformation of MDCK cells increases DAG levels, thereby activating and, in turn, down-regulating PK-C and certain responses to TPA. Since activation of PK-C may result in a variety of effects on signal transduction pathways, we propose that increased DAG and altered PK-C levels associated with ras transformation may account for the inconsistent effects previously observed in studies evaluating the effect of ras transformation on phospholipases and other signal transduction systems.

Differential activation of protein kinase C alpha is associated with arachidonate release in Madin-Darby canine kidney cells.

The heterogeneity of the protein kinase C (PKC) gene family strongly suggests that different isoforms may have distinct functions in mediating signal transduction. However, there is very little direct evidence for this. PKC has been implicated in arachidonate (AA) release in many cell types. We sought to investigate whether bradykinin- and phorbol ester-stimulated AA release in Madin-Darby canine kidney (MDCK) cells was correlated with differential activation of PKC isoforms. Using phorbol esters to (i) activate the enzyme and (ii) to down-regulate it, we report that differential activation (translocation) of PKC alpha is associated with AA release in MDCK cells and that specific down-regulation of PKC alpha is associated with a loss of AA release in response to stimulation with dioctanoylglycerol and phorbol ester. We also demonstrate that bradykinin-stimulated AA release was associated with differential activation of PKC alpha and was inhibited in PKC alpha down-regulated cells. Thus, we conclude that the PKC alpha isoform is likely to be responsible for mediating AA release in these cells.

Inhibition of expression of protein kinase C alpha by antisense cDNA inhibits phorbol ester-mediated arachidonate release.

A major unresolved issue in the area of signal transduction relates to the role of particular isoforms of protein kinase C (PKC) in mediating cellular responses subsequent to activation of that enzyme. We have addressed this issue by the use of antisense technology. We have stably transfected Madin-Darby canine kidney cells with antisense PKC alpha, PKC beta, or both PKC alpha and -beta cDNAs. The transfected cDNA was integrated and expressed. We have isolated cells in which expression of PKC alpha is inhibited. In cells transfected with antisense PKC alpha or both PKC alpha and -beta, phorbol ester-stimulated release of arachidonate and its metabolites was inhibited, whereas in cells transfected with antisense PKC beta cDNA alone, phorbol ester-stimulated arachidonate release was not significantly different from control cells. We thus demonstrate the use of a novel technique to inhibit PKC isoform expression. We show that inhibition of expression of PKC alpha causes a loss in phospholipase A2-mediated arachidonate release. Antisense-inhibited expression of PKC isoforms may provide a useful approach to define additional functions of particular PKC isoforms.