Pro-resolving lipid mediators: Agents of anti-ageing?

Inflammation is an essential response to injury and its timely and adequate resolution permits tissue repair and avoidance of chronic inflammation. Ageing is associated with increased inflammation, sub-optimal resolution and these act as drivers for a number of ageing-associated pathologies. We describe the role played by specialised proresolving lipid mediators (SPMs) in the resolution of inflammation and how insufficient levels of these mediators, or compromised responsiveness may play a role in the pathogenesis of many ageing-associated pathologies, e.g. Alzheimer's Disease, atherosclerosis, obesity, diabetes and kidney disease. Detailed examination of the resolution phase of inflammation highlights the potential to harness these lipid mediators and or mimetics of their bioactions, in particular, their synthetic analogues to promote effective resolution of inflammation, without compromising the host immune system.

New susceptibility loci associated with kidney disease in type 1 diabetes.

Diabetic kidney disease, or diabetic nephropathy (DN), is a major complication of diabetes and the leading cause of end-stage renal disease (ESRD) that requires dialysis treatment or kidney transplantation. In addition to the decrease in the quality of life, DN accounts for a large proportion of the excess mortality associated with type 1 diabetes (T1D). Whereas the degree of glycemia plays a pivotal role in DN, a subset of individuals with poorly controlled T1D do not develop DN. Furthermore, strong familial aggregation supports genetic susceptibility to DN. However, the genes and the molecular mechanisms behind the disease remain poorly understood, and current therapeutic strategies rarely result in reversal of DN. In the GEnetics of Nephropathy: an International Effort (GENIE) consortium, we have undertaken a meta-analysis of genome-wide association studies (GWAS) of T1D DN comprising ~2.4 million single nucleotide polymorphisms (SNPs) imputed in 6,691 individuals. After additional genotyping of 41 top ranked SNPs representing 24 independent signals in 5,873 individuals, combined meta-analysis revealed association of two SNPs with ESRD: rs7583877 in the AFF3 gene (P = 1.2 × 10(-8)) and an intergenic SNP on chromosome 15q26 between the genes RGMA and MCTP2, rs12437854 (P = 2.0 × 10(-9)). Functional data suggest that AFF3 influences renal tubule fibrosis via the transforming growth factor-beta (TGF-ß1) pathway. The strongest association with DN as a primary phenotype was seen for an intronic SNP in the ERBB4 gene (rs7588550, P = 2.1 × 10(-7)), a gene with type 2 diabetes DN differential expression and in the same intron as a variant with cis-eQTL expression of ERBB4. All these detected associations represent new signals in the pathogenesis of DN.

A novel hybrid CFH/CFHR3 gene generated by a microhomology-mediated deletion in familial atypical hemolytic uremic syndrome.

Genomic disorders affecting the genes encoding factor H (fH) and the 5 factor H related proteins have been described in association with atypical hemolytic uremic syndrome. These include deletions of CFHR3, CFHR1, and CFHR4 in association with fH autoantibodies and the formation of a hybrid CFH/CFHR1 gene. These occur through nonallelic homologous recombination secondary to the presence of large segmental duplications (macrohomology) in this region. Using multiplex ligation-dependent probe amplification to screen for such genomic disorders, we have identified a large atypical hemolytic uremic syndrome family where a deletion has occurred through microhomology-mediated end joining rather than nonallelic homologous recombination. In the 3 affected persons of this family, we have shown that the deletion results in formation of a CFH/CFHR3 gene. We have shown that the protein product of this is a 24 SCR protein that is secreted with normal fluid-phase activity but marked loss of complement regulation at cell surfaces despite increased heparin binding. In this study, we have therefore shown that microhomology in this area of chromosome 1 predisposes to disease associated genomic disorders and that the complement regulatory function of fH at the cell surface is critically dependent on the structural integrity of the whole molecule.

Lipoxins attenuate renal fibrosis by inducing let-7c and suppressing TGFßR1.

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA4) attenuated TGF-ß1-induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-ß1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-ß1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA4 analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-ß1 signaling pathway, including the TGF-ß receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-ß receptor type 1 and the response to TGF-ß1. Overexpression of let-7c mimicked the antifibrotic effects of LXA4 in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA4. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA4-mediated upregulation of let-7c suppresses TGF-ß1-induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.

Chromosome 2q31.1 associates with ESRD in women with type 1 diabetes.

Sex and genetic variation influence the risk of developing diabetic nephropathy and ESRD in patients with type 1 diabetes. We performed a genome-wide association study in a cohort of 3652 patients from the Finnish Diabetic Nephropathy (FinnDiane) Study with type 1 diabetes to determine whether sex-specific genetic risk factors for ESRD exist. A common variant, rs4972593 on chromosome 2q31.1, was associated with ESRD in women (P<5×10(-8)) but not in men (P=0.77). This association was replicated in the meta-analysis of three independent type 1 diabetes cohorts (P=0.02) and remained significant for women (P<5×10(-8); odds ratio, 1.81 [95% confidence interval, 1.47 to 2.24]) upon combined meta-analysis of the discovery and replication cohorts. rs4972593 is located between the genes that code for the Sp3 transcription factor, which interacts directly with estrogen receptor α and regulates the expression of genes linked to glomerular function and the pathogenesis of nephropathy, and the CDCA7 transcription factor, which regulates cell proliferation. Further examination revealed potential transcription factor-binding sites within rs4972593 and predicted eight estrogen-responsive elements within 5 kb of this locus. Moreover, we found sex-specific differences in the glomerular expression levels of SP3 (P=0.004). Overall, these results suggest that rs4972593 is a sex-specific genetic variant associated with ESRD in patients with type 1 diabetes and may underlie the sex-specific protection against ESRD.

The dual role of lipids in chronic kidney disease: Pathogenic culprits and therapeutic allies.

Chronic kidney disease (CKD) is a significant health burden, with rising incidence and prevalence, attributed in part to increasing obesity and diabetes rates. Lipid accumulation in the kidney parenchyma and chronic, low-grade inflammation are believed to significantly contribute to the development and progression of CKD. The effect of dysregulated kidney lipid metabolism in CKD progression, including altered cholesterol and fatty acid metabolism contribute to glomerular and tubular cell injury through the activation of oxidative stress and inflammatory signalling cascades. In contrast, classes of endogenous specialized pro-resolving lipid mediators (SPMs) have been described that act to limit the inflammatory response and promote the resolution of inflammation. This review highlights our current understanding of how lipids can cause damage within the kidney, and classes of protective lipid metabolites that offer therapeutic benefits.

Next-generation sequencing identifies TGF-ß1-associated gene expression profiles in renal epithelial cells reiterated in human diabetic nephropathy.

Transforming growth factor-beta (TGF-ß1) is implicated in the onset and progression of renal fibrosis and diabetic nephropathy (DN), leading to a loss of epithelial characteristics of tubular cells. The transcriptional profile of renal tubular epithelial cells stimulated with TGF-ß1 was assessed using RNA-Seq, with 2027 differentially expressed genes identified. Promoter analysis of transcription factor binding sites in the TGF-ß1 responsive gene set predicted activation of multiple transcriptional networks, including NFκB. Comparison of RNA-Seq with microarray data from identical experimental conditions identified low abundance transcripts exclusive to RNA-Seq data. We compared these findings to human disease by analyzing transcriptomic data from renal biopsies of patients with DN versus control groups, identifying a shared subset of 179 regulated genes. ARK5, encoding an AMP-related kinase, and TGFBI - encoding transforming growth factor, beta-induced protein were induced by TGF-ß1 and also upregulated in human DN. Suppression of ARK5 attenuated fibrotic responses of renal epithelia to TGF-ß1 exposure; and silencing of TGFBI induced expression of the epithelial cell marker - E-cadherin. We identified low abundance transcripts in sequence data and validated expression levels of several transcripts (ANKRD56, ENTPD8) in tubular enriched kidney biopsies of DN patients versus living donors. In conclusion, we have defined a TGF-ß1-driven pro-fibrotic signal in renal epithelial cells that is also evident in the DN renal transcriptome.

The effect of bariatric surgery on renal function and disease: a focus on outcomes and inflammation.

Renal dysfunction and disease, including hyperfiltration, proteinuria and hypofiltration, are commonly associated with obesity. Diabetic kidney disease is also common in obese cohorts. Weight loss interventions, including bariatric surgery, can effectively reduce weight and improve renal outcomes. Some of this effect may be due to the remission of Type 2 diabetes and hypertension. However, other mechanisms, including the resolution of inflammatory processes, may also contribute. The effect of bariatric surgery on renal function has only recently become a focus of particular investigation. In this study, we will review the effects of bariatric surgery on obesity-associated kidney disease. We will discuss the pitfalls in assessing renal function in obese cohorts and will examine the effect of bariatric surgery on renal function and urinary protein excretion using different mechanisms. We will give particular attention to the evidence for bariatric surgery in cohorts with established renal disease and suggest future directions. In particular, we will outline the evidence for inflammation as an important therapeutic target, and the emerging medical therapies being considered to exploit this target in obesity- and diabetes-related kidney disease.

Epigenome-wide meta-analysis identifies DNA methylation biomarkers associated with diabetic kidney disease.

Type 1 diabetes affects over nine million individuals globally, with approximately 40% developing diabetic kidney disease. Emerging evidence suggests that epigenetic alterations, such as DNA methylation, are involved in diabetic kidney disease. Here we assess differences in blood-derived genome-wide DNA methylation associated with diabetic kidney disease in 1304 carefully characterised individuals with type 1 diabetes and known renal status from two cohorts in the United Kingdom-Republic of Ireland and Finland. In the meta-analysis, we identify 32 differentially methylated CpGs in diabetic kidney disease in type 1 diabetes, 18 of which are located within genes differentially expressed in kidneys or correlated with pathological traits in diabetic kidney disease. We show that methylation at 21 of the 32 CpGs predict the development of kidney failure, extending the knowledge and potentially identifying individuals at greater risk for diabetic kidney disease in type 1 diabetes.

A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer.

BACKGROUND: NET1, a RhoA guanine exchange factor, is up-regulated in gastric cancer (GC) tissue and drives the invasive phenotype of this disease. In this study, we aimed to determine the role of NET1 in GC by monitoring the proliferation, motility and invasion of GC cells in which NET1 has been stably knocked down. Additionally, we aimed to determine NET1-dependent transcriptomic events that occur in GC. METHODS: An in vitro model of stable knockdown of NET1 was achieved in AGS human gastric adenocarcinoma cells via lentiviral mediated transduction of short-hairpin (sh) RNA targeting NET1. Knockdown was assessed using quantitative PCR. Cell proliferation was assessed using an MTS assay and cell migration was assessed using a wound healing scratch assay. Cell invasion was assessed using a transwell matrigel invasion assay. Gene expression profiles were examined using affymetrix oligonucleotide U133A expression arrays. A student's t test was used to determine changes of statistical significance. RESULTS: GC cells were transduced with NET1 shRNA resulting in a 97% reduction in NET1 mRNA (p < 0.0001). NET1 knockdown significantly reduced the invasion and migration of GC cells by 94% (p < 0.05) and 24% (p < 0.001) respectively, while cell proliferation was not significantly altered following NET1 knockdown. Microarray analysis was performed on non-target and knockdown cell lines, treated with and without 10 µM lysophosphatidic acid (LPA) allowing us to identify NET1-dependent, LPA-dependent and NET1-mediated LPA-induced gene transcription. Differential gene expression was confirmed by quantitative PCR. Shortlisted NET1-dependent genes included STAT1, TSPAN1, TGFBi and CCL5 all of which were downregulatd upon NET1 downregulation. Shortlisted LPA-dependent genes included EGFR and PPARD where EGFR was upregulated and PPARD was downregulated upon LPA stimulation. Shortlisted NET1 and LPA dependent genes included IGFR1 and PIP5K3. These LPA induced genes were downregulated in NET1 knockdown cells. CONCLUSIONS: NET1 plays an important role in GC cell migration and invasion, key aspects of GC progression. Furthermore, the gene expression profile further elucidates the molecular mechanisms underpinning NET1-mediated aggressive GC cell behaviour.

A GREM1 gene variant associates with diabetic nephropathy.

Gremlin, a cell growth and differentiation factor, promotes the development of diabetic nephropathy in animal models, but whether GREM1 gene variants associate with diabetic nephropathy is unknown. We comprehensively screened the 5' upstream region (including the predicted promoter), all exons, intron-exon boundaries, complete untranslated regions, and the 3' region downstream of the GREM1 gene. We identified 31 unique variants, including 24 with a minor allele frequency exceeding 5%, and 9 haplotype-tagging single nucleotide polymorphisms (htSNPs). We selected one additional variant that we predicted to alter transcription factor binding. We genotyped 709 individuals with type 1 diabetes of whom 267 had nephropathy (cases) and 442 had no evidence of kidney disease (controls). Three individual SNPs significantly associated with nephropathy at the 5% level, and two remained significant after adjustment for multiple testing. Subsequently, we genotyped a replicate population comprising 597 cases and 502 controls: this population supported an association with one of the SNPs (rs1129456; P = 0.0003). Combined analysis, adjusted for recruitment center (n = 8), suggested that the T allele conferred greater odds of nephropathy (OR 1.69; 95% CI 1.36 to 2.11). In summary, the GREM1 variant rs1129456 associates with diabetic nephropathy, perhaps explaining some of the genetic susceptibility to this condition.

Rapid and Laboratory SARS-CoV-2 Antibody Testing in High-Risk Hospital Associated Cohorts of Unknown COVID-19 Exposure, a Validation and Epidemiological Study After the First Wave of the Pandemic.

Objective: We aimed to use SARS-CoV-2 antibody tests to assess the asymptomatic seroprevalence of individuals in high-risk hospital cohorts who's previous COVID-19 exposure is unknown; staff, and patients requiring haemodialysis or chemotherapy after the first wave. Methods: In a single Center, study participants had five SARS-CoV-2 antibody tests done simultaneously; one rapid diagnostic test (RDT) (Superbio Colloidal Gold IgM/IgG), and four laboratory tests (Roche Elecsys® Anti-SARS-CoV-2 IgG [RE], Abbott Architect i2000SR IgG [AAr], Abbott Alinity IgG [AAl], and Abbott Architect IgM CMIA). To determine seroprevalence, only positive test results on laboratory assay were considered true positives. Results: There were 157 participants, of whom 103 (65.6%) were female with a median age of 50 years (range 19-90). The IgG component of the RDT showed a high number of false positives (n = 18), was inferior to the laboratory assays (p < 0.001 RDT vs. AAl/AAr, p < 0.001 RDT vs. RE), and had reduced specificity (85.5% vs. AAl/AAr, 87.2% vs. RE). Sero-concordance was 97.5% between IgG laboratory assays (RE vs. AAl/AAr). Specificity of the IgM component of the RDT compared to Abbott IgM CMIA was 95.4%. Ten participants had positivity in at least one laboratory assay, seven (9.9%) of which were seen in HCWs. Two (4.1%) hematology/oncology (H/O) patients and a single (2.7%) haemodialysis (HD) were asymptomatically seropositive. Asymptomatic seroprevalence of HCWs compared to patients was not significant (p = 0.105). Conclusion: HCWs (9.9%) had higher, although non-significant asymptomatic seroprevalence of SARS-CoV-2 antibodies compared to high-risk patients (H/O 4.1%, HD 2.7%). An IgM/IgG rapid diagnostic test was inferior to laboratory assays. Sero-concordance of 97.5% was found between IgG laboratory assays, RE vs. AAl/AAr.

Resequencing of the CCL5 and CCR5 genes and investigation of variants for association with diabetic nephropathy.

Chemokine (C-C motif) ligand 5 (CCL5) and chemokine (C-C motif) receptor 5 are implicated in the pathogenesis of diabetic nephropathy (DN). We hypothesize that variants in these genes may be associated with DN. The CCL5 and chemokine receptor type 5 (CCR5) genes were resequenced, variants identified (n=58), allele frequencies determined in 46 individuals (92 chromosomes) and efficient haplotype tag single-nucleotide polymorphisms (htSNPs) selected to effectively evaluate the common variation in these genes. One reportedly functional gene variant and eight htSNPs were genotyped in a case-control association study involving Caucasian individuals with type 1 diabetes (267 cases with DN and 442 non-nephropathic diabetic controls). Genotyping was performed using MassARRAY iPLEX, TaqMan, gel electrophoresis and direct capillary sequencing. After correction for multiple testing, there were no statistically significant associations between variants in the CCL5 and CCR5 genes and DN.

A rare haplotype of the vitamin D receptor gene is protective against diabetic nephropathy.

BACKGROUND: Vitamin D and its analogues are reported to have renoprotective effects in chronic kidney disease including diabetic nephropathy (DN). Vitamin D(3) is converted to 1,25(OH)D(3) by CYP2R1 and CYP27B1. The biological action of 1,25(OH)D(3) is mediated via its receptor. VDR, CYP27B1 or CYP2R1 gene variants could modify the biological activity of vitamin D(3). We have conducted the first case-control association study to determine the relationship between polymorphisms in VDR, CYP27B1 and CYP2R1 genes, and the risk of DN in individuals with type 1 diabetes. METHODS: Eight VDR single-nucleotide polymorphisms (SNPs) rs10735810 FokI C>T, rs1544410 BsmI G>A, rs7975232 ApaI G>T, rs731236 TaqI T>C, rs4303288 G>T, rs11168275 C>T, rs12721366 G>A and rs2544043 G>C were investigated with CYP27B1 rs4646536 T>C and CYP2R1 rs10741657 G>A. Genotyping was performed using pyrosequencing, Taqman, Sequenom or direct sequencing technologies in 1329 type 1 diabetics (655 nephropaths, 674 non-nephropaths). RESULTS: No significant differences were observed in genotype or allele frequencies between case and control groups for VDR, CYP27B1 or CYP2R1 SNPs, either before or after stratification by recruitment centre or when restricted to patients with end-stage renal disease. A previously identified haplotype block from rs1544410 to rs731236 was confirmed at the 3'-end of VDR. Comparison of haplotype frequencies identified the rare AGT haplotype as significantly protective against DN, 3.1% cases versus 5.8% controls; chi(2) = 11.05, Pc = 0.009 by the permutation test. CONCLUSIONS: Our study has identified a rare VDR haplotype that is protective against DN in patients with type 1 diabetes. Replication in a large, independent cohort is required to confirm this finding.

Diagnostic utility of genetic testing in patients undergoing renal biopsy.

High-throughput DNA testing is becoming established as a standard diagnostic test in the renal clinic. Previously published studies on cohorts of patients with unexplained chronic kidney disease of a suspected genetic aetiology have suggested a diagnostic yield for genomic sequencing of up to 18%. Here we determine the yield of targeted gene panel in a clinically unscreened cohort of patients referred for percutaneous native renal biopsy. Patients who underwent renal biopsy for investigation of chronic kidney disease were sequenced using a genomic sequencing panel covering 227 genes in which variation is known to be associated with monogenic chronic kidney disease (CKD). Candidate disease-causing variants were assessed for pathogenicity using guidelines from the American College for Medical Genetics and Genomics. Fifty CKD patients were recruited and sequenced. A molecular diagnosis was obtained for two patients (4%). A molecular diagnosis is possible using genomic testing in ∼4% of clinically unscreened patients undergoing renal biopsy. Genetic screening may be useful for diagnosis in a subset of CKD patients but is most valuable when applied to patients with suspected heritable forms of kidney disease.

Genetic analysis of coronary artery disease single-nucleotide polymorphisms in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is a leading cause of end-stage renal disease. Premature mortality is common in patients with nephropathy, largely due to cardiovascular disease. Genetic variants implicated in macrovascular disease are therefore excellent candidates to assess for association with diabetic nephropathy. Recent genome-wide association studies have identified a total of 15 single-nucleotide polymorphisms (SNPs) that are reproducibly associated with cardiovascular disease. METHODS: We initially assessed these SNPs for association in UK type 1 diabetic patients with (cases; n = 597) and without (controls; n = 502) nephropathy using iPLEX(TM) and TaqMan(R) assays. Replication studies were performed with DNA genotyped in a total of 2668 individuals from the British Isles. RESULTS: One SNP (rs4420638) on chromosome 19q13 was found to be significantly associated with diabetic nephropathy before (P = 0.0002) and after correction for multiple testing (P(corrected) = 0.002). We replicated this finding in a phenotypically similar case-control collection comprising 709 individuals with type 1 diabetes (P = 0.002; combined P < 0.00001; OR = 1.54, 95% CI: 1.29-1.84). CONCLUSIONS: Our case-control data suggest that rs4420638, or a functional SNP in linkage disequilibrium with this SNP, may be associated with diabetic nephropathy.

Resequencing of genes for transforming growth factor beta1 (TGFB1) type 1 and 2 receptors (TGFBR1, TGFBR2), and association analysis of variants with diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is the leading cause of end stage renal failure in the western world. There is substantial epidemiological evidence supporting a genetic predisposition to diabetic nephropathy, however the exact molecular mechanisms remain unknown. Transforming growth factor (TGFbeta1) is a crucial mediator in the pathogenesis of diabetic nephropathy. METHODS: We investigated the role of five known single nucleotide polymorphisms (SNPs) in the TGFB1 gene for their association with diabetic nephropathy in an Irish, type 1 diabetic case (n = 272) control (n = 367) collection. The activity of TGFbeta1 is facilitated by the action of type 1 and type 2 receptors, with both receptor genes (TGFBR1 and TGFBR2) shown to be upregulated in diabetic kidney disease. We therefore screened TGFBR1 and TGFBR2 genes for genomic variants using WAVEtrade mark (dHPLC) technology and confirmed variants by direct capillary sequencing. Allele frequencies were determined in forty-eight healthy individuals. Data for all SNPs was assessed for Hardy Weinberg equilibrium, with genotypes and allele frequencies compared using the chi2 test for contingency tables. Patterns of linkage disequilibrium were established and common haplotypes estimated. RESULTS: Fifteen variants were identified in these genes, seven of which are novel, and putatively functional SNPs were subsequently genotyped using TaqMantrade mark, Invadertrade mark or Pyrosequencing(R) technology. No significant differences (p > 0.1) were found in genotype or allele distributions between cases and controls for any of the SNPs assessed. CONCLUSION: Our results suggest common variants in TGFB1, TGFBR1 and TGFBR2 genes do not strongly influence genetic susceptibility to diabetic nephropathy in an Irish Caucasian population.

Gene expression analysis in human osteoblasts exposed to dexamethasone identifies altered developmental pathways as putative drivers of osteoporosis.

BACKGROUND: Osteoporosis, a disease of decreased bone mineral density represents a significant and growing burden in the western world. Aging population structure and therapeutic use of glucocorticoids have contributed in no small way to the increase in the incidence of this disease. Despite substantial investigative efforts over the last number of years the exact molecular mechanism underpinning the initiation and progression of osteoporosis remain to be elucidated. This has meant that no significant advances in therapeutic strategies have emerged, with joint replacement surgery being the mainstay of treatment. METHODS: In this study we have used an integrated genomics profiling and computational biology based strategy to identify the key osteoblast genes and gene clusters whose expression is altered in response to dexamethasone exposure. Primary human osteoblasts were exposed to dexamethasone in vitro and microarray based transcriptome profiling completed. RESULTS: These studies identified approximately 500 osteoblast genes whose expression was altered. Functional characterization of the transcriptome identified developmental networks as being reactivated with 106 development associated genes found to be differentially regulated. Pathway reconstruction revealed coordinate alteration of members of the WNT signaling pathway, including frizzled-2, frizzled-7, DKK1 and WNT5B, whose differential expression in this setting was confirmed by real time PCR. CONCLUSION: The WNT pathway is a key regulator of skeletogenesis as well as differentiation of bone cells. Reactivation of this pathway may lead to altered osteoblast activity resulting in decreased bone mineral density, the pathological hallmark of osteoporosis. The data herein lend weight to the hypothesis that alterations in developmental pathways drive the initiation and progression of osteoporosis.

Microarray identifies ADAM family members as key responders to TGF-beta1 in alveolar epithelial cells.

The molecular mechanisms of Idiopathic Pulmonary Fibrosis (IPF) remain elusive. Transforming Growth Factor beta 1(TGF-beta1) is a key effector cytokine in the development of lung fibrosis. We used microarray and computational biology strategies to identify genes whose expression is significantly altered in alveolar epithelial cells (A549) in response to TGF-beta1, IL-4 and IL-13 and Epstein Barr virus. A549 cells were exposed to 10 ng/ml TGF-beta1, IL-4 and IL-13 at serial time points. Total RNA was used for hybridisation to Affymetrix Human Genome U133A microarrays. Each in vitro time-point was studied in duplicate and an average RMA value computed. Expression data for each time point was compared to control and a signal log ratio of 0.6 or greater taken to identify significant differential regulation. Using normalised RMA values and unsupervised Average Linkage Hierarchical Cluster Analysis, a list of 312 extracellular matrix (ECM) proteins or modulators of matrix turnover was curated via Onto-Compare and Gene-Ontology (GO) databases for baited cluster analysis of ECM associated genes. Interrogation of the dataset using ontological classification focused cluster analysis revealed coordinate differential expression of a large cohort of extracellular matrix associated genes. Of this grouping members of the ADAM (A disintegrin and Metalloproteinase domain containing) family of genes were differentially expressed. ADAM gene expression was also identified in EBV infected A549 cells as well as IL-13 and IL-4 stimulated cells. We probed pathologenomic activities (activation and functional activity) of ADAM19 and ADAMTS9 using siRNA and collagen assays. Knockdown of these genes resulted in diminished production of collagen in A549 cells exposed to TGF-beta1, suggesting a potential role for these molecules in ECM accumulation in IPF.

Expression of gremlin, a bone morphogenetic protein antagonist, in human diabetic nephropathy.

BACKGROUND: We report the induction of gremlin, a bone morphogenetic protein antagonist, in cultured human mesangial cells exposed to high glucose and transforming growth factor beta (TGF-beta) levels in vitro and kidneys from diabetic rats in vivo. METHODS: Gremlin expression was assessed in human diabetic nephropathy by means of in situ hybridization, immunohistochemistry, and real-time polymerase chain reaction and correlated with clinical and pathological indices of disease. RESULTS: Gremlin was not expressed in normal human adult kidneys. Conversely, abundant gremlin expression was observed in human diabetic nephropathy. Although some gremlin expression was observed in occasional glomeruli, gremlin expression was most prominent in areas of tubulointerstitial fibrosis, where it colocalized with TGF-beta expression. Gremlin messenger RNA levels correlated directly with renal dysfunction, determined by means of serum creatinine level, but not with proteinuria level. There was a strong correlation between gremlin expression and tubulointerstitial fibrosis score. CONCLUSION: In aggregate, these results indicate that the developmental gene gremlin reemerges in the context of tubulointerstitial fibrosis in diabetic nephropathy and suggests a role for TFG-beta as an inducer of gremlin expression in this context.